Recent improvements in electron microscopes in equipment for digital image processing imaging processing, and in methods for biological specimen preparation and analysis have set the stage for a revolution in cellular electron microscopy. This Program Project assembles cell biologists at the University of Colorado with diverse scientific and technological experience so they can make a contribution to this will be significant. Our program project includes a major section on Core Technological Research and Development in which we will implement highly efficient EM tomography of significant volumes from fast frozen, freeze-substituted cells whose preservation for structural study represents the state-of-the art. Core projects include novel approaches to the labeling of specific cellular macromolecules, innovations in structural modeling technology, and new approaches to the labeling of specific cellular macromolecules, innovations in structural modeling technology, and new approaches to thinking about the structural variability of organelles. Three scientific projects will capitalize on this technology and contribute to its development: an analysis of Golgi structure and function in mammalian cells; descriptions of Golgi variability and complexity in yeast, algae, and plants; a structural dissection of the centrosome from budding yeast; and an experimental/descriptive analysis of mammalian kinetochores. Each of these projects will require the instrumentation and technology developed in the Core, but at the same time each scientific project will provide a viable test bed for the efficacy of the new technologies developed. The members of each project are accustomed to collaborative effort, and they see this integrated approach to improving the study of cellular structure as a major project for their coming 5 years of research.