Vinflunine, a newly synthesized derivative, possesses marked in vivo antitumor properties and, like other alkaloids, inhibits in vitro tubulin assembly at microM concentrations. However, in contrast to other vinca alkaloids, vinflunine exhibits relatively low in vitro cytotoxic potency. The aim of this report was to investigate whether the action(s) of vinflunine on the microtubule cytoskeleton could account for its cytotoxicity or if its cellular action requires another molecular target. Four vinca alkaloids used in cancer therapy and vinflunine were studied using PtK2 cells. Their activities on the most dynamic microtubules were investigated in mitosis and in interphase by evaluating the disturbance of the metaphase plate and the splitting of the diplosome, respectively. No correlation was observed between the cellular accumulation of these compounds and either their cytotoxicity or their action(s) on the microtubule cytoskeleton. In contrast, cytotoxicity, mitotic disturbance and diplosome splitting were observed in the nM range for vinblastine, vincristine, vindesine and vinorelbine, although these events occurred at 10 times higher concentrations in the case of vinflunine. Hence, dynamic modifications of both the mitotic and interphasic microtubule cytoskeleton are compatible with in vitro cytotoxicity of vinflunine, raising questions about the conventional biochemical screening of these vinca alkaloids.