Vinblastine, a highly successful antitumor drug, targets the tubulin molecule. Tubulin, the subunit protein of microtubules, consists of an alpha- and a beta-subunit, both of which consist of isotypes encoded by different genes. We have purified three isotypes of bovine brain tubulin, namely, alpha(beta)II, alpha(beta)III and alpha(beta)IV. Microtubule associated protein-2 (MAP2) and Tau-induced assembly of these isotypes were compared in the presence and absence of vinblastine. MAP2-induced assembly of unfractionated tubulin and all the isotypes except alpha(beta)II tubulin was resistant to 1 microM vinblastine. Vinblastine at low concentrations (< 10 microM) progressively inhibited the assembly of all of the isotypes but the vinblastine concentration required for inhibition of MAP2-induced microtubule assembly was minimal for alpha(beta)II. The tau-induced assembly of unfractionated tubulin and alpha(beta)III were equally sensitive to 1 microM vinblastine whereas alpha(beta)II and alpha(beta)IV were much more sensitive to vinblastine. The microtubules obtained in the presence of tau from unfractionated tubulin, alpha(beta)II and alpha(beta)IV could be easily aggregated by 20 microM vinblastine whereas such as aggregation of microtubules obtained from alpha(beta)III and tau required approximatedly 40 microM vinblastine. Our results suggest that among the tubulin isotypes, alpha(beta)II is the most sensitive to vinblastine in the presence of MAPs while alpha(beta)III is the most resistant and this intrinsic resistance of alpha(beta)III dimers persists in the polymeric form of alpha(beta)III tubulin as well. These results may be relevant to the therapeutic and toxic actions of vinblastine.