Tumor vasculature offers a target for drugs directed against proliferating endothelia. We hypothesized that cucurbitacin E (CuE), an actin-disrupting agent, would preferentially inhibit proliferating vs. quiescent endothelia. Log-phase and confluent ECV304 and HUVEC human endothelial cells were exposed to 10-200 nM CuE for 1-96 hr, and toxicity was determined at 2-6 days by sulforhodamine B assay. Confluent ECV cells were scraped by Q-tip to allow proliferation at the margin and exposed to CuE at LD50, and the actin cytoskeleton was stained by rhodamine-phalloidin. Cell cycle analysis was performed by flow cytometry. Log-phase cells were significantly inhibited compared to confluent. LD50's of log-phase cells were much less than for confluent cells (12 vs. 170 nM, ECV; 13 vs 76 nM, HUVEC). Persistent growth inhibition required 24-96 hr exposure to LD50. Followed less than 6 hr exposure. CuE induced F-actin to accumulate centrally in clumps in newly proliferating cells; actin appeared normal in quiescent cells. CuE treated endothelial cells were not blocked at cytokinesis. CuE preferentially potently inhibits proliferating human endothelia compared to quiescent cells in vitro. CuE induces depolymerization of F-actin in proliferating cells. At low concentrations, cucurbitacin E may potently inhibit vascular proliferation by disrupting actin.