The NIH Roadmap Initiative has as one of its key underlying objectives the implementation of high through put, multiplexed instruments for application in a wide variety of basic and clinical research. The flow cytometry platform is ideally suited to this purpose, and several recent technologies have significantly increased throughput and simultaneous, multiparametric analysis. We propose to provide several critical technology advances by extending the capabilities and applications of flow-based analysis and sorting to produce an increasingly multiparameter, high-throughput analysis and screening tool. This will be accomplished through an integrated plan of three Research and Development Projects, extensive internal and external Collaborations, a wide variety of Service projects, expansion of our Training efforts, and increased Dissemination activities. The R&D Projects aim to extend flow cytometry into three areas of direct relevance to the Roadmap goals: 1) application of acoustic focusing and sensing technology to provide both novel analysis parameters and in-line sample preparation;2) increasing both the throughput and the particle size range of flow sorting technologies;and 3) developing an integrated instrument to collect complete emission spectra separated according to fluorescence lifetime. Each of these projects has a defined set of collaborations that apply the new technologies to a wide range of biomedical research, including protein engineering, protein-protein interactions, drug and ligand screening, multiplexed cellular analysis, and automated preparation and analysis of complex clinical and environmental samples. In addition, we will continue and expand our Collaborative projects using instruments developed in the previous funding period. We will expand our Service efforts by providing access to a wide variety of commercial and unique developmental instruments. We will continue our traditionally outstanding Training program of hands-on courses and student/PostDoc mentorship, and develop new courses focused on application of our recent technology advances. Our strong Dissemination program will also be extended to include an improved website and the sponsorship of three focused meetings on various aspects of advanced flow cytometry. In conclusion, we propose to continue to drive the design, development, and application of flow cytometry technology as a multiplexed, high-throughput platform for advanced biomedical and clinical research.