The in vitro effects of four Vinca alkaloids, vinblastine (VLB), vincristine (VCR), vindesine (VDS) and vinepidine (VPD), on B16 melanoma proliferation, binding to bovine brain tubulin and B16 melanoma cell extracts, and uptake by the B16 cells were compared. The relative binding affinities to bovine brain tubulin were VPD greater than VCR congruent to VDS greater than VLB with the Ka for VPD being about 4-fold higher than that for VLB. On the other hand, the relative effects on B16 cell proliferation were exactly the opposite. Differences were found in the degree of concentration of the four alkaloids by the cells: 100-fold for VLB, 50-fold for VCR and VDS, and 20-fold for VPD. At the extracellular concentrations of drugs which inhibit proliferation by 50%, the intracellular concentration would still be far less than the tubulin concentration. Thus, it is likely that all of the Vinca alkaloids would be bound to tubulin and difference in uptake rather than Ka values is the major factor in determining the relative effectiveness of the drugs. L cells showed 50% the sensitivity of B16 melanoma cells toward VLB and 30% the sensitivity toward VPD. The L cells also concentrated these drugs to a lesser extent than did the B16 cells.