Studies have shown salutary effects of 17β-estradiol following traumahemorrhage on different cell types. 17β-Estradiol also induces improved circulation via relaxation of the aorta and has an anti-apoptotic effect on endothelial cells. Because mitochondria play a pivotal role in apoptosis, we hypothesized that 17β-estradiol will maintain mitochondrial function and will have protective effects against H2O 2-induced apoptosis in endothelial cells. Endothelial cells were isolated from rats' aorta and cultured in the presence or absence of H 2O2, a potent inducer of apoptosis. In additional studies, endothelial cells were pretreated with 17β-estradiol. Flow cytometry analysis revealed H2O2-induced apoptosis in 80.9% of endothelial cells; however, prior treatment of endothelial cells with 17β-estradiol resulted in an ∼40% reduction in apoptosis. This protective effect of 17β-estradiol was abrogated when endothelial cells were cultured in the presence ICI-182780, indicating the involvement of estrogen receptor (ER). Fluorescence microscopy revealed a 17β-estradiol-mediated attenuation of H2O2-induced mitochondrial condensation. Western blot analysis demonstrated that H2O2-induced cytochrome c release from mitochondrion to cytosol and the activation of caspase-9 and -3 were decreased by 17β-estradiol. These findings suggest that 17β-estradiol attenuated H2O2-induced apoptosis via ER-dependent activation of caspase-9 and -3 in rat endothelial cells through mitochondria. Copyright © 2007 the American Physiological Society.