b-Lapachone [b-lap; 3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione] is a novel anticancer drug currently under investigation in phase I/II clinical trials. However, the mechanism underlying its clinical efficacy remains unclear. In this study, we found that b-lap provoked the cleavage of heat shock protein 90 (Hsp90) in NAD(P)H:quinone oxidoreductase-1 (NQO1)-expressing lung and prostate cancer cells as well as in primary human umbilical vein endothelial cells (HUVECs). These actions of b-lap were different from that of the conventional Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin. As a consequence of Hsp90 cleavage, Hsp90-associated oncoproteins, such as receptor-interacting protein, Raf-1, AKT, and CDK4, were degraded in treated cancer cells, and key receptor tyrosine kinases such as vascular endothelial cell growth factor receptor-2 and Her-2 were degraded in treated HUVECs through a proteasomal system. Further results revealed that specific inhibitors of NQO1 and reactive oxygen species could dramatically reduce b-lap-mediated Hsp90 cleavage. In addition to its cytotoxicity, b-lap effectively inhibited angiogenesis by suppressing tube formation and the invasion of HUVECs in vitro, rat aortic microvascular sprouts ex vivo, and mouse corneal neovascularization in vivo. Furthermore, b-lap markedly suppressed the growth and angiogenesis of human lung cancer xenografts in nude mice and decreased the levels of receptorinteracting protein, AKT, CDK4, and CD31 in the solid tumors. Unlike other NQO1-dependent cytotoxic quinones, such as streptonigrin, menadione, mitomycin, and 17-allylamino-17-demethoxygeldanamycin, b-lap was the only agent that could cause Hsp90 cleavage. Taken together, our results suggest a crucial mechanism underlying the antitumor efficacy of b-lap.