Vinblastine is an antimitotic drug that inhibits microtubule assembly and induces the self-association of tubulin into coiled spiral aggregates. Previous quantitative binding and sedimentation velocity results have been interpreted by a mechanism involving isodesmic ligand-mediated plus ligand-facilitated self-association [Na, G., & Timasheff, S. N. (1986) Biochemistry 25, 6214-6222, 6222-6228]. In this study, the vinblastine-induced self-association of porcine brain tubulin has been compared in the presence of 50 microM GDP or 50 microM GTP to investigate the role of nucleotides. Experiments at 1-4 microM tubulin in 10 mM Pipes, 1 mM MgSO4, 2 mM EGTA (pH 6.9), and varying concentrations of vinblastine (0.05-70 microM) demonstrate that GDP enhances self-association by 2-4-fold over GTP. In the presence of GDP or GTP, sedimentation velocity data can be best fit by either an indefinite ligand-mediated model or an indefinite ligand-mediated plus ligand-facilitated model. The association constant, K2, for the vinblastine-tubulin complex binding to a polymer is larger when GDP is present, while the association constant, K1, for the binding of vinblastine to tubulin heterodimers is identical in the presence of either nucleotide. The enhancement of K2 by GDP is confirmed by micropartition binding experiments with [3H]vinblastine. The fitting of sedimentation velocity and binding studies gives parameters for the interaction of vinblastine with GTP-tubulin that are identical, within error, to the previous results of Na and Timasheff. van't Hoff analysis of multiple temperature data reveals that this enhancement in the presence of GDP is due to a change in the enthalpy of self-association. Additional results suggest that the interaction of vinblastine with tubulin is identical for all beta-isotypes. Sedimentation velocity experiments in the presence of GDP or GTP show that the vinblastine-induced association of affinity-purified alpha,beta-class III tubulin is identical to that of unfractionated tubulin, although there is a difference in the abilities of unfractionated tubulin and alpha,beta III-tubulin to associate into taxol-stabilized microtubules.