Estrogen causes nitric oxide (NO)-dependent vasodilation due to estrogen receptor (ER) &agr;-mediated, nongenomic activation of endothelial NO synthase (eNOS). The subcellular site of interaction between ER&agr; and eNOS was determined in studies of isolated endothelial cell plasma membranes. Estradiol (E2, 10–8mol/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 ER&agr; 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 ER&agr; displayed ER-mediated eNOS stimulation, whereas membranes from cells expressing eNOS alone or ER&agr; plus a myristoylation-deficient mutant eNOS were insensitive. Fractionation of endothelial cell plasma membranes revealed ER&agr; protein in caveolae, and E2caused 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 E2on eNOS in caveolae was prevented by calcium chelation. Thus, a subpopulation of ER&agr; 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.