FR901228, a natural cyclic depsipeptide, shows high cytotoxicity against human cancer cell lines (low nM IC50 values). Cells exposed to FR901228 arrest with G1 or G2/M DNA content; S phase is depleted. G2/M cells include cells arrested in mitosis. We wished to understand the mitotic arrest by this compound. Mitotic arrest is often due to interference with microtubules and COMPARE testing in the NCI drug screen indicated a possible taxane-like mechanism. Testing of FR901228 for tubulin binding or alteration of in vitro MT assembly failed to reveal any effect. Likewise, examination of cellular microtubules following exposure to FR901228 did not reveal any change. Similar G2/M accumulation was observed in MCF7, MCF10 and PC3 cells. About 50% of G2/M cells were mitotic and contained microtubule spindles. Mitotic cells peaked at about 14-16 h drug exposure and declined to near 0% by 24-30 h. The block was at prometaphase, with numerous chromosomes unattached to the spindle. We conclude that FR901228 induces formation of aberrant spindles probably by interfering with chromosome attachment, causing mitotic accumulation without affecting mitotic microtubules.