We have investigated the cell cycle inhibition mechanism and primary target of tryprostatin A (TPS-A) purified from Aspergillus fumigatus. TPS-A inhibited cell cycle progression of asynchronously cultured 3Y1 cells in the M phase in a dose- and time-dependent manner. In contrast, TPS-B (the demethoxy analogue of TPS-A) showed cell-cycle non-specific inhibition on cell growth even though it inhibited cell growth at lower concentrations than TPS-A. TPS-A treatment induced the reversible disruption of the cytoplasmic microtubules of 3Y1 cells as observed by indirect immunofluorescence microscopy in the range of concentrations that specifically inhibited M-phase progression. TPS-A inhibited the assembly in vitro of microtubules purified from bovine brains (40% inhibition at 250 µM); however, there was little or no effect on the self-assembly of purified tubulin when polymerization was induced by glutamate even at 250 µM TPS-A. TPS-A did not inhibit assembly promoted by taxol or by digestion of the C-terminal domain of tubulin. However, TPS-A blocked the tubulin assembly induced by inducers interacting with the C-terminal domain, microtubule-associated protein 2 (MAP2), tau and poly-(l-lysine). These results indicate that TPS-A is a novel inhibitor of MAP-dependent microtubule assembly and, through the disruption of the microtubule spindle, specifically inhibits cell cycle progression at the M phase.