The tubulin-binding agent combretastatin A-4-phosphate (CA-4-P), rapidly disrupts the vascular network of tumors leading to secondary tumor cell death. In vitro, CA-4-P destabilizes microtubules and causes endothelial cell death. In this study we analyze the mechanisms by which CA-4-P induces the death of proliferating endothelial cells. We demonstrate that at >/=7.5 nmol/L, CA-4-P damages mitotic spindles, arrests cells at metaphase, and leads to the death of mitotic cells with characteristic G(2)/M DNA content. Mitotic arrest was associated with elevated levels of cyclin B1 protein and p34(cdc2) activity. Inhibition of p34(cdc2) activity by purvalanol A caused mitotic-arrested cells to rapidly exit mitosis, suggesting that sustained p34(cdc2) activity was responsible for metaphase arrest. Pharmacological prevention of entry into mitosis protected cells from undergoing cell death, further establishing the link between mitosis and cell death induction by CA-4-P. CA-4-P-mediated cell death shared characteristics of apoptosis but was independent of caspase activation suggesting the involvement of a non-caspase pathway(s). These data suggest that induction of apoptosis in endothelial cells by CA-4-P is associated with prolonged mitotic arrest. Therefore, by activating cell death pathways, CA-4-P, in addition to being an effective anti-vascular agent, may also interfere with regrowth of blood vessels in the tumor.