—Estrogen causes nitric oxide (NO)-dependent vasodilation due to estrogen receptor (ER) α-mediated, nongenomic activation of endothelial NO synthase (eNOS). The subcellular site of interaction between ERα and eNOS was determined in studies of isolated endothelial cell plasma membranes. Estradiol (E₂, 10^–8 mol/L) caused an increase in eNOS activity in plasma membranes in the absence of added calcium, calmodulin, or eNOS cofactors, which was blocked by ICI 182,780 and ERα antibody. Immunoidentification studies detected the same 67-kDa protein in endothelial cell nucleus, cytosol, and plasma membrane. Plasma membranes from COS-7 cells expressing eNOS and ERα displayed ER-mediated eNOS stimulation, whereas membranes from cells expressing eNOS alone or ERα plus a myristoylation-deficient mutant eNOS were insensitive. Fractionation of endothelial cell plasma membranes revealed ERα protein in caveolae, and E₂ caused stimulation of eNOS in isolated caveolae that was ER-dependent; noncaveolae membranes were insensitive. Acetylcholine and bradykinin also activated eNOS in isolated caveolae. Furthermore, the effect of E₂ on eNOS in caveolae was prevented by calcium chelation. Thus, a subpopulation of ERα is localized to endothelial cell caveolae where they are coupled to eNOS in a functional signaling module that may regulate the local calcium environment. The full text of this article is available at http://www.circresaha.org.