The Kinesin-2 family of molecular motors transport cargo along cilia and flagella in a process termed intraflagellar transport. Disruption of intraflagellar transport results in truncated cilia and flagella at the cellular level, and at the organismal level leads to defects in body plan development and organ failure. There is a concerted effort underway to define the molecular machinery underlying intraflagellar transport, but characterizing this process at the molecular scale is made difficult by the virtually complete lack of understanding of the molecular mechanism of the Kinesin-2 motor. To answer cellular questions such as how the motor activity is regulated and how many motors are required for transport, it is essential to first characterize Kinesin-2 motors at the single-molecule level, and define the key biochemical transitions that underlie their mechanism. A unique and puzzling feature of Kinesin-2 motors is that instead of containing two identical motor domains like most kinesins, they are made up of two different motor domains. Domain swapping experiments carried out in the PI's lab have now shown that these two heads move at different speeds, leading to the hypothesis that their activities are tuned to optimize transport characteristics of the intact heterodimer. The goal of the proposed work is to define the molecular mechanism of Kinesin-2 function by characterizing KIF3A/B, the mouse Kinesin-2 ortholog. Single-molecule fluorescence and optical tweezer experiments will measure the performance characteristics of these motors (speed, force production and microtubule affinity) and will uncover the inner workings of these motors to reveal the structural basis of movement and regulation. The specific aims are as follows: 1) Test whether the two heads step along microtubules at different rates. 2) Determine whether kinetic differences arise from structural differences in the motor domains or from coordination between the motor domains. 3) Measure the number of sequential steps motors take during each encounter with a microtubule and the dependence of this processivity on external load. 4) Identify the biochemical transitions in the Kinesin-2 kinetic cycle that control motor speed and processivity. Experimental data will be incorporated into computational models of Kinesin-2 motility to test hypotheses regarding the cellular behavior of these motors. Understanding the molecular mechanism of Kinesin-2 motility is important for uncovering the molecular basis of transport-based diseases such as polycystic kidney disease, defects in sperm motility, and retinal degeneration. Furthermore, insights into the mechanochemistry of kinesin motors will aid in developing anti- tumor therapies targeting mitotic kinesins.