INTRODUCTIONImportant advances in our understanding of the organization and dynamics of the cytoskeleton have been made by direct observations of fluorescently tagged cytoskeletal proteins in living cells. In early experiments, the cytoskeletal protein of interest was purified, covalently modified with a fluorescent dye, and microinjected into living cells. In the mid-1990s, a powerful new technology arose: Researchers developed methods for expressing chimeric proteins consisting of the gene of interest fused to green fluorescent protein (GFP). This approach has become a standard method for characterizing protein localization and dynamics. More recently, a profusion of “XFP” (spectral variants of GFP) has been developed, allowing researchers straightforwardly to perform experiments ranging from simultaneous co-observation of protein dynamics to fluorescence recovery after photobleaching (FRAP), fluorescence resonance energy transfer (FRET), and subresolution techniques such as stimulated emission-depletion microscopy (STED) and photoactivated localization microscopy (PALM). In this article, the methods used to express and analyze GFP- and/or XFP-tagged tubulin and microtubule-associated proteins (MAPs) are discussed. Although some details may be system-specific, the methods and considerations outlined here can be adapted to a wide variety of proteins and organisms.