Microtubules are engaged in many important activities in eukaryotic cells. They form the scaffolding that helps determine overall cell shape, as well as the arrangement of interior organelles. Intracellular communication and the traffic of vesicles depend upon the microtubule network. In dividing cells, the microtubules cycle between the generalized multifunctional interphase network that provides access to every corner of the cell, and a mitotic apparatus that is centrally located and specialized for the task of separating chromosomes. As would be expected for such a heavily used object, agents that interfere with microtubule function strike at the heart of many cellular activities and often have profound physiological effects. Some of these agents have therapeutic applications in humans: colchicine, used for its anti-inflammatory properties in gouty arthritis; vincristine, vinblastine, and taxol, used for their anti-mitotic effects against a variety of neoplasms; griseofulvin, an anti-fungal agent; and others. Besides these medicinal uses, agents that affect microtubules are significant for human health in equally important, though non-therapeutic ways, such as their widespread use as pesticides and fungicides. Basic information about microtubules therefore has an unusually direct and immediate connection to the mission of the NIH. As expected from their diverse activities, microtubules interact with many other cellular proteins. To understand these interactions in a way that will allow rational manipulation of microtubule-based phenomena, which would certainly be a potent therapeutic and diagnostic capability, we must know the structure of microtubules in great detail. We must also know the structure of the supramolecular assemblies that use microtubules to provide the motion-producing, shape-determining, and organelle-positioning functions within cells. Fortunately, and remarkably, the basic structure of the microtubule seems to be constant regardless of the particular assembly in which it is involved. Thus if we can determine the structure of any one of the functional classes of microtubule, we will have learned a lot about all microtubules. This proposal describes our successful preparation of native cellular microtubules suitable for structure determination by cryo-electron microscopy of unfixed, unstained, frozen hydrated samples. We report some preliminary results from examining these microtubules by cryo-electron microscopy, and propose a set of experiments that will yield a high resolution 3D structure of the native microtubule.