The interaction of nocodazole with calf brain tubulin was studied to determine the effect of such interaction on the structure of tubulin. The effect of nocodazole on the self-association of tubulin was monitored by turbidity measurements and velocity sedimentation. Sedimentation patterns indicate that nocodazole neither induces tubulin to undergo self-association to form higher orders of aggregate nor does it perturb the equilibrium of the reaction leading to the formation of 42S double-ring structures although nocodazole binds to both the tubulin dimers and the polymeric form. Nocodazole does, however, inhibit the in vitro reconstitution of microtubules, and the presence of microtubule-associated proteins does not amplify the inhibitory effect of the drug. The conformational changes in tubulin upon binding of nocodazole were monitored by differential spectroscopy, circular dichroism, fluorescence, and chemical modification of sulfhydryl residues. Results from these studies show that the sulfhydryl residues become more accessible to chemical modification. In contrast, the binding of nocodazole does not significantly alter the net environment of tryptophan chromophores. These residues are apparently not all located on the surface of the tubulin molecule and at least some are partially buried.