—17β-Estradiol (E₂) is a rapid activator of endothelial nitric oxide synthase (eNOS). The product of this activation event, NO, is a fundamental determinant of cardiovascular homeostasis. We previously demonstrated that E₂-stimulated endothelial NO release can occur without an increase in cytosolic Ca²⁺. Here we demonstrate for the first time, to our knowledge, that E₂ rapidly induces phosphorylation and activation of eNOS through the phosphatidylinositol 3 (PI3)-kinase–Akt pathway. E₂ treatment (10 ng/mL) of the human endothelial cell line, EA.hy926, resulted in increased NO production, which was abrogated by the PI3-kinase inhibitor, LY294002, and the estrogen receptor antagonist ICI 182,780. E₂ stimulated rapid Akt phosphorylation on serine 473. As has been shown for vascular endothelial growth factor, eNOS is an E₂-activated Akt substrate, demonstrated by rapid eNOS phosphorylation on serine 1177, a critical residue for eNOS activation and enhanced sensitivity to resting cellular Ca²⁺ levels. Adenoviral-mediated EA.hy926 transduction confirmed functional involvement of Akt, because a kinase-deficient, dominant-negative Akt abolished E₂-stimulated NO release. The membrane-impermeant E₂BSA conjugate, shown to bind endothelial cell membrane sites, also induced rapid Akt and consequent eNOS phosphorylation. Thus, engagement of membrane estrogen receptors results in rapid endothelial NO release through a PI3-kinase–Akt-dependent pathway. This explains, in part, the reduced requirement for cytosolic Ca²⁺ fluxes and describes an important pathway relevant to cardiovascular pathophysiology.