Matrix ligation of integrins alphavbeta3/alphavbeta5 is critical for endothelial survival and angiogenesis. We have previously shown that ceramide, a proapoptotic lipid second messenger, increases during endothelial anoikis (detachment-induced apoptosis). We now show that RGDfV, an integrin alphavbeta3/alphavbeta5 cyclic function-blocking peptide, increased ceramide and decreased sphingomyelin in human brain microvascular endothelial cells (HBMECs) plated on vitronectin, suggesting that sphingomyelin hydrolysis contributes to RGDfV-induced ceramide increase. Desipramine and imipramine, inhibitors of acid sphingomyelinase (ASMase), suppressed RGDfV-induced ceramide increase. Importantly, desipramine, imipramine, and a third ASMase inhibitor, SR33557, but not inhibitors of neutral sphingomyelinase, suppressed RGDfV-induced apoptosis, suggesting that ASMase was required for integrin-mediated apoptosis. Myriocin, an inhibitor of de novo ceramide synthesis, had no effect on RGDfV-induced HBMEC apoptosis. Interestingly, ASMase inhibitors also suppressed the RGDfV-induced loss of spreading on vitronectin. RGDfV induced a similar increase in ceramide and apoptosis in HBMECs on poly-l-lysine or vitronectin, although cells detached only from vitronectin, indicating that cell detachment was not required for RGDfV-induced apoptosis. Our results suggest involvement of ASMase and ceramide in endothelial apoptosis induced by inhibition of integrins alphavbeta3/alphavbeta5, and propose a novel molecular mechanism for the antiangiogenic effect of RGDfV.