Estradiol retards the development of atherosclerosis. Animal models have suggested that NO may be a critical effector molecule in this cardiovascular protection. In this study, female human umbilical vein endothelial cells (HUVECs) were propagated in phenol red–free gonadal hormone–free medium and pretreated with 17β-estradiol (E₂). Reduced NO₂⁻ and NO₃⁻ (NO_X) concentration, determined by chemiluminescence, demonstrated a rapid increase in basal HUVEC NO release in response to physiological concentrations of E₂. The estrogen receptor (ER) antagonist ICI 164,384 inhibited the augmented NO release, demonstrating an ER-mediated component of this response. Because endothelial NO synthase (eNOS) activity is largely regulated by cytosolic Ca²⁺, relative [Ca²⁺]_i in response to E₂ was determined in a fluorometric assay. E₂ did not promote HUVEC Ca²⁺ fluxes. Furthermore, eNOS activity in E₂-pretreated endothelial whole-cell lysates was not dependent on additional Ca²⁺. Despite involving the ER, this is a nongenomic effect of E₂, as demonstrated by maintained responses in transcriptionally inhibited cells and by the rapidity (10 minutes) of cGMP formation in an NO bioassay. We demonstrate, for the first time, that independent of cytosolic Ca²⁺ mobilization, there is augmentation of eNOS activity with a resultant increase in HUVEC basal NO release in response to short-term estradiol exposure. Implications for the cardiovascular protective role of estrogen are discussed.