Estrogen protects against the development of coronary heart disease in women. This study was designed to examine the direct effects of estrogen on nitric oxide release and endothelial nitric oxide synthase (eNOS) expression in cultured human coronary artery endothelial cells (HCAECs). NOx (nitrate, nitrite, and nitric oxide) was measured by the chemiluminescence method. Prolonged treatment (48 h) of the cells with 17β-estradiol (E2β), but not 17α-estradiol (E2α), resulted in a 2.3-fold increase in basal NOx release in HCAECs and an enhanced adenosine triphosphate (ATP)- and calcium ionophore A23187-induced NOx release. The effects of E2β on endothelial NOx release were blocked by estrogen-receptor antagonist ICI 182,780. E2β had no effect on basal and ATP-stimulated intracellular Ca2+ concentrations in HCAECs. However, E2β significantly increased eNOS protein levels, as determined by Western analysis. We conclude that estrogen increases NOx release in HCAECs, which is independent of cytosolic Ca2+ mobilization and is mediated by the upregulation of eNOS.