Vinblastine binding to tublin was measured in different buffers using tubulin prepared by two different methods and three different binding assay methods. In 100 mM 1,4-piperazinediethanesulfonic acid (Pipes) buffer containing 1 mM MgSO4 and 1 mM ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), the data appeared to be consistent with one site with a Ka value of 3.4 X 10(6) M-1 and another site with a Ka value of 2.8 X 10(5) M-1. However, in buffers of lower ionic strengths and without Mg2+ the Ka values were lower. The lowest value (2 X 10(4) M-1) was obtained in 10 mM phosphate buffer, in which only one site was evident under the conditions used. Neither the binding assay used nor the method for tubulin preparation affected the Ka value. Using HPLC, aggregation induced by vinblastine was evident in buffers which gave the largest Ka values. Tubulin aggregation in the presence of vinblastine was also confirmed by analytical ultracentrifugation. The results support the proposal of Na and Timasheff [Biochemistry 25, 6214 (1986)] that the apparent Ka value is influenced by the degree of aggregation induced by vinblastine and that the intrinsic binding constant to the dimer is represented by the lowest value, about 2 X 10(4) M-1.