Studies have shown salutary effects of 17-estradiol following trauma-hemorrhage on different cell types. 17-estradiol also induces improved circulation via relaxation of aorta and has an anti-apoptotic effect on endothelial cells. Since mitochondria play a pivotal role in apoptosis, we hypothesized that 17-estradiol will maintain mitochondrial function and will have protective effects against H₂O₂-induced apoptosis in endothelial cells. Endothelial cells were isolated from rat aorta and cultured in the presence or absence of H₂O₂, a potent inducer of apoptosis. In additional studies, endothelial cells were pretreated with 17-estradiol. Flow cytometry analysis revealed H₂O₂-induced apoptosis in 80.9% of endothelial cells; however, prior treatment of endothelial cells with 17-estradiol resulted in an approximately 40% reduction in apoptosis. This protective effect of 17-estradiol was abrogated when endothelial cells were cultured in the presence ICI-182780, indicating the involvement of ER. Fluorescence microscopy revealed a 17-estradiol-mediated attenuation of H₂O₂-induced mitochondrial condensation. Western blot analysis demonstrated that H₂O₂-induced cytochrome c release from mitochondrion to cytosol as well as the activation of caspase-9 and -3 were decreased by 17-estradiol. These findings suggest that 17-estradiol attenuated H₂O₂-induced apoptosis via ER-dependent activation of caspase-9 and -3 in rat endothelial cells through mitochondria.