Mitostatic action, cellular uptake and the binding of colchicine and colcemid to tubulin were compared. It was shown that mitostatic action of low doses of colchicine developed only after 24 h incubation of the drug with mouse L fibroblasts, while the colcemid-induced block of mitosis was evident after 2 h incubation. The initial rate of uptake was about 10 times greater for colcemid than for colchicine. Cellular uptake of the drugs reached an equilibrium after 2 and 15-18 h incubation for colcemid and colchicine, respectively, and the plateau values were identical. The kinetics of colchicine and colcemid binding to bovine brain tubulin was studied by the DEAE-filter binding assay. Colcemid binds to tubulin much faster than does colchicine. The rate of colcemid efflux from L cells is much higher than that of colchicine. According to the efflux data, colcemid dissociates readily from a complex with tubulin (t1/2 = 10 min), while the colchicine-tubulin complex is stable for at least 1 h. These results are consistent with previously published data (Frankel, F.R. (1976) Proc. Natl. Acad. Sci. U.S.A. 72, 2798-2802), which showed that colcemid action on cells is more reversible than that of colchicine. We suggest that differences between colchicine and colcemid in the rate of mitostatic action and its reversibility are determined by the differences in parameters of tubulin binding.