Background A series of studies was performed to examine the ability of estradiol (E ₂ ) to protect endothelial cells from apoptosis. Methods and Results Light and transmission electron microscopy demonstrated typical features of apoptosis in human umbilical vein endothelial cells (HUVEC) exposed to tumor necrosis factor-α (TNF-α). Northern and Western blot analyses revealed induction of message and protein for the interleukin-1β converting enzyme (ICE), which has been shown to mediate apoptosis induced by TNF-α. Immunofluorescent staining of HUVEC colocalized ICE expression to apoptotic HUVEC. Direct cell counting demonstrated a significant decrease in total endothelial cell number after 24 hours of TNF-α exposure and a dose-dependent reversal of the effect of TNF-α with E ₂ treatment. This protective effect was abrogated by an estrogen-receptor antagonist. Fluorescence-activated cell sorting analysis revealed 39.3% apoptosis after 24 hours of TNF-α exposure. Treatment with E ₂ resulted in a 50% decrease in apoptosis. Similarly, viability assays revealed 35±4% cell death after TNF-α exposure. Simultaneous treatment with E ₂ resulted in a dose-dependent reduction of cell death to a minimum of 18±2%. The protective effect of E ₂ was nullified by a specific estrogen-receptor antagonist. Conclusions E ₂ treatment resulted in a dose-dependent, receptor-mediated inhibition of TNF-α–induced endothelial cell apoptosis. These studies indicate that E ₂ may also serve a maintenance function in preventing endothelial cell death after noxious stimuli and suggest that the ICE pathway may mediate cytokine-induced apoptosis in endothelial cells. Preservation of endothelial integrity represents another mechanism that may account for the atheroprotective effect of estrogen.