Cemadotin (LU103793) (NSC D-669356) is a water-soluble synthetic analogue of dolastatin 15 that inhibits cell proliferation in vitro and the growth of human tumor xenografts. Cemadotin is in phase II clinical trials as a promising cancer chemotherapeutic agent. The drug blocks cells at mitosis. Its primary mode of action has been unclear but is believed to involve an action on microtubules. We have found that cemadotin binds to tubulin and strongly suppresses microtubule dynamics. Scatchard analysis of cemadotin binding to tubulin indicated that there are two affinity classes of cemadotin-binding sites with Kd values of 19.4 microM and 136 microM. Cemadotin did not inhibit the binding of vinblastine to tubulin, and, conversely, vinblastine did not inhibit the binding of cemadotin to tubulin. By quantitative video microscopy of individual microtubules, we found that cemadotin strongly suppressed dynamic instability of microtubules assembled to steady state using bovine brain tubulin devoid of microtubule-associated proteins. It reduced the rate and extent of growing and shortening, increased the rescue frequency, and increased the percentage of time the microtubules spent in an attenuated or paused state, neither growing nor shortening detectably. At the lowest effective cemadotin concentrations, dynamics were suppressed in the absence of significant microtubule depolymerization. The results suggest that cemadotin exerts its antitumor activity by suppressing spindle microtubule dynamics through a distinct molecular mechanism by binding at a novel site in tubulin.