Vinflunine is a novel fluorinated Vinca alkaloid currently in Phase II clinical trials, which in preclinical studies exhibited superior antitumor activity to that of two clinically useful Vinca alkaloids, vinorelbine and vinblastine. All three of the drugs block mitosis at the metaphase/anaphase transition, leading to apoptosis. The mechanism of the mitotic block is not known. On the basis of results with purified microtubules and in living interphase cells, we hypothesized that it involves suppression of spindle microtubule dynamics. Here we measured the effects of the three Vinca alkaloids on dynamics of centromeres and spindle kinetochore-microtubules by a novel approach involving quantitative time-lapse confocal microscopy in living mitotic human U2OS cells. Green fluorescent protein-labeled centromere-binding protein B was used to mark centromeres and kinetochore-microtubule plus ends. In controls, pairs of centromeres on sister chromatids alternated under tension between increasing and decreasing separation (stretching and relaxing). All three of the Vinca alkaloids suppressed centromere dynamics similarly at concentrations that block mitosis. At concentrations approximating the IC(50)s for mitotic accumulation (18.8 nM vinflunine, 7.3 nM vinorelbine, and 6.1 nM vinblastine), centromere dynamicity decreased by 44%, 25%, and 26%, respectively, and the time centromeres spent in a paused state increased by 63%, 52%, and 36%, respectively. Centromere relaxation rates, stretching durations, and transition frequencies all decreased. Thus all three of the drugs decreased the normal microtubule-dependent spindle tension at the centromeres/kinetochores, thereby preventing the signal for mitotic checkpoint passage. The strong correlation between suppression of kinetochore-microtubule dynamics and mitotic block indicates that the primary mechanism by which the Vinca alkaloids block mitosis is suppression of spindle microtubule dynamics.