A competitive inhibitor of colchicine binding to tubulin has been found in rat brain. Most of the inhibitor is associated with microsomes but some inhibitor, with an apparent molecular weight of approximately 250,000, is found in the cytosol. Both the microsomal and cytosol inhibitors are heat- and trypsin-sensitive, indicating that a protein moiety is required for activity. The microsomes bind tubulin directly; the microsomal and cytosol fractions both inhibit microtubule assembly in vitro. The inhibitor may function in the living cell to bind and sequester non-polymerized tubulin. Regulation of tubulin attachment to microsomes could then control the concentration of cytosolic tubulin available for microtubule assembly.