Up until FY 2008, the ID09B time-resolved X-ray beamline at the European Synchrotron and Radiation Facility (ESRF) in Grenoble, France was the only facility in the world capable of acquiring time-resolved macromolecular structures with 150-ps time resolution and <2-Angstrom spatial resolution. The Anfinrud group was instrumental in helping develop that capability. Unfortunately, the ESRF operates in a mode that is optimized for time-resolved Laue crystallography studies only 14 days out of each year, and we had access to only a portion of this limited amount of beam time. To expand the amount of beam time available for our studies, we partnered with the Advanced Photon Source (APS) in Argonne, IL and BioCARS to develop picosecond time-resolved X-ray capabilities on Sector 14 at the APS. BioCARS is an NIH-funded beamline headed by Prof. Keith Moffat, and is operated by the University of Chicago. In FY2005, Dr. Marvin Gershengorn, then Director of Intramural Research at NIDDK, committed >$1M to procure the capital equipment needed for this effort. Our vision was to achieve picosecond time-resolved X-ray capabilities comparable to that realized at the ESRF when the APS is operated in 24-bunch mode, a common operating mode used 132 days per year. This goal required that we isolate a single bunch of X-rays from a train of pulses separated by only 153 ns, a feat that we first achieved in July 2007 using a high-speed chopper, whose rotor was fabricated according to our design. Because the quality of time-resolved X-ray diffraction data is ultimately limited by the number of photons delivered to the sample in a single X-ray pulse, it was crucial to achieve X-ray fluence comparable to that generated at the ESRF when they operate in their exotic 4-bunch mode. To that end, we replaced the existing U33 undulator (33-mm magnetic period) with two newly designed U23 and U27 undulators, making BioCARS the first APS beamline to operate with two inline undulators. NIDDK funded this effort, with the APS supplying the labor to design and refurbish two undulators according to our performance specifications. When the gaps of these undulators are tuned to generate 12-keV X-ray photons, the X-ray fluence is comparable to that generated at the ESRF during their 4-bunch mode. When the APS operates in their exotic hybrid mode, which is scheduled approximately 31 days per year, the X-ray fluence is a factor of 4 higher than that available with the ESRF 4-bunch mode. These achievements increase by more than an order of magnitude the amount of beamtime available worldwide to pursue 100 ps time-resolved X-ray science. The infrastructure needed to pursue picosecond time-resolved X-ray studies goes far beyond delivering single X-ray pulses to the experimental hutch. We installed a picosecond laser system in a laser hutch located near the X-ray hutch as well as an array of laser diagnostics that aid in the optimization of the laser performance. We have also developed a Field-Programmable-Gate-Array (FPGA) based timing system that synchronizes all time-critical components to the X-ray pulses. For example, the FPGA drives the heat-load chopper, the high-speed chopper, the picosecond laser system, a millisecond shutter, and various other motion controls that must be synchronized with the x-ray pulse arrival time. Importantly, we can set the time delay between X-ray and laser pulses from picoseconds to seconds with a precision of 10 ps. We also developed the diffractometer used to acquire time-resolved X-ray diffraction images. This effort included the design and fabrication of a millisecond shutter, a motorized support for the high-speed X-ray chopper, a support for motorized X-ray slits, detectors for non-invasively monitoring the laser and X-ray pulse energy and relative time delay, a motorized stage for the X-ray detector, supports for a collimator pipe and X-ray beam stop, beam conditioning optics that tailor the laser pulses in both space and time, beam delivery optics that focus the laser pulses onto the sample, motorized controls to center the focused laser pulse on the sample, and motorized controls to center the collimator pipe on the X-ray beam. Finally, we continue to refine the software required to control the beamline. This software package, called LaueCollect, is written in the Python programming language, and is generalized for both time-resolved Laue crystallography and time-resolved SAXS/WAXS studies. For these experiments, it is crucial to achieve long-term stability of the focused spot position, a challenging proposition due to the large distance between the laser system and the sample (>30 m). Thermal drift in the laser hutch, the hall of the synchrotron ring, and the X-ray hutch affect the beam position, and can cause the focus spot to drift from its set point. To mitigate this problem, we have developed a protocol to monitor and maintain the laser alignment. We installed a camera to monitor the beam position within the beam conditioning optics enclosure and a second camera to image the focused spot at the sample position. If the beam position drifts from the cross hairs of either camera by more than a preset threshold, the corresponding beam-steering optics are adjusted using precision motors and the beam is re-centered. This combination of controls allows us to maintain precise overlap between the laser and X-ray pulses in the sample. Time-resolved X-ray studies require precise overlap of the laser and X-ray pulses in both space and time. Due to the large mismatch in the laser and X-ray penetration depths in protein crystals, we employ an orthogonal pump-probe geometry with the laser beam directed downward and the X-ray beam horizontal. It is crucial that the top edge of the protein crystal be positioned at the intersection of the two beams. If the crystal height is set too low, we observe no diffraction;if set too high, the laser pulse cannot penetrate to the depth of the X-ray pulse, and we observe no pump-induced changes in the crystal diffraction. We have developed an improved method for efficiently and reliably finding the top edge of the crystal. With motion controls integrated into our microscope camera software, we can define visually the edge of the crystal in three-dimensional space by pointing and clicking along the top edge of the crystal, a process that is repeated at various phi angles. This definition of the crystal edge is approximate, and is used as a starting point for determining the edge of the crystal using X-rays, which is much more precise. To minimize radiation damage when executing the edge finding algorithm, we acquire diffraction images from 10-fold attenuated X-ray pulses. We scan the crystal vertically in 20 &#956;m steps (half the vertical dimension of the X-ray beam) and determine the integrated spot intensity from a single X-ray pulse at each position. We developed an efficient algorithm for quantifying the integrated intensity of spots on the detector, with each step of the scan taking less than 0.5 s. The integrated spot intensity is proportional to the crystal volume intercepted by the X-ray pulse, and is used to define the edge of the crystal. Once found, the crystal is raised 40 &#956;m to position the top edge of the crystal at the top edge of the X-ray beam. This procedure ensures precise positioning of the crystal, and has demonstrably improved the quality of the time-resolved diffraction data.