The antimitotic drugs colchicine, podophyllotoxin, and vinblastine are known to be potent inhibitors of microtubule polymerization, but little is known about how they affect the chemical properties of the tubulin molecule. In the preceding paper [Ludueña, R. F., & Roach, M. C. (1981) Bio-chemistry (preceding paper in this issue)], we have shown that the alkylating agent iodo[14C]acetamide reacts specifically with the sulfhydryl groups of tubulin and that its bifunctional analogue, N,N'-ethylenebis(iodoacetamide) (EBI), reacts with native tubulin to convert beta-tubulin into a form, designated beta*, which appears to represent an intrachain cross-linked form of beta. In this paper, we have incubated tubulin with the drugs prior to alkylation and measured their effects on the alkylation reactions. We have found that at 100 microM concentrations, podophyllotoxin, colchicine, and vinblastine inhibited the reaction of tubulin with iodo[14C]acetamide by 19-32%, 33-47%, and 62-72%, respectively; each drug was half-maximally effective at 3-5 microM, indicating that the suppressive effects of the drugs were mediated by their high-affinity binding sites. Similarly, beta* formation induced by EBI was suppressed by 92-94% in the presence of either colchicine or podophyllotoxin In contrast, vinblastine enhanced beta* formation by 40%. Alkylation with longer chain analogues of EBI revealed no evidence that the reactive sulfhydryls were being pushed apart by the drugs. These results indicate that each of the drugs has potent effects on the accessibility of the sulfhydryl groups of tubulin and that the effects of vinblastine are very different from those of either colchicine or podophyllotoxin.