A novel potent protein factor capable of inhibiting the in vitro polymerization of mammalian brain microtubule protein and of breaking down preformed microtubules has been partially purified from cell extracts of Dictyostelium discoideum. The factor has an apparent Mr of around 13000 and is trypsin resistant but heat and pepsin sensitive. When soluble microtubule protein was fractionated into tubulin and microtubule-associated proteins and each fraction was assayed independently for its susceptibility toward inhibition, it was clearly demonstrated that the tubulin but not the associated protein fraction was rendered nonpolymerizable. Soluble tubulin was inactivated at ratios of 1 mol of inhibitor to 100 mol of tubulin, estimated conservatively. Quantitative separation of tubulin and inhibitor after inactivation did not result in reactivation of tubulin's polymerizing capacity, suggesting a catalytic modification. The biochemical properties tested of the inactive tubulin argue against a mechanism involving simple proteolysis, N-site GTP hydrolysis or release, or general denaturation.