Vascular homeostasis is largely governed by regulation of endothelial nitric oxide synthase (eNOS), and the consequent elaboration of nitric oxide (NO). NO creates an anti-platelet aggregatory, anti-leukocyte adhesive and anti-smooth muscle cell proliferative environment. As such, the healthy endothelium is self-protective, limiting the initiation and progression of atherosclerosis. Although considerable debate exists regarding the cardiovascular benefits of hormone replacement therapy in post-menopausal women (1), there is an extensive body of molecular and animal model data demonstrating that estrogen activates eNOS through rapid, receptor-mediated posttranslational modifications. It is thus imperative, for the purpose of highly specific therapeutic targeting, to understand the biology of estrogen receptors (ERs) in the endothelium. This includes defining predominant ER isoforms, subcellular localization and targeting, protein-protein interactions and, ultimately, 3-dimensional structure. We have shown that the predominant ER alpha isoform in endothelial cells (ECs) from multiple vascular beds, in multiple species, is a 46 kDa splice variant, devoid of the N-terminal AF-1 domain (2). A subset of these receptors, expressed in ECs, is targeted to specialized lipid rafts, caveolae, at the plasma membrane (3). This membrane targeting is dependent on receptor palmitoylation and interactions with membrane-targeted c-Src, a non-receptor tyrosine kinase, the function of which is critical for ER46-mediated rapid estrogen responses. In ex vivo studies, aortic rings from c-Src gene-deleted mice lose their endothelium-dependent vasodilating responses to estrogen (4). Fluorescence Resonance Energy Transfer imaging demonstrates a direct ER46-c-Src protein-protein interaction, enhanced by E2. Membrane-impermeant forms of 17 beta-estradiol (E2) cause endothelial c-Src, phosphatidylinositol 3-kinase, Akt and eNOS activation within 20 minutes (5). Furthermore, E2 stimulates dynamic and rapid recruitment of more ERs to the plasma membrane, thereby amplifying the response to available ligand. These features of ER expression and rapid activation are also seen in human endothelial progenitor cells. ER46 contains a highly conserved hydrophobic sequence (IILL 450-453) that is also critical for productive insertion into the plasma membrane. When mutated to hydrophilic residues, membrane localization, impermeant ligand binding and rapid signaling responses are impaired. Although considerable crosstalk between membrane- and nuclear-initiated (genomic) responses occurs, it is important to define those features of endothelial membrane ERs to which selective therapeutic agents could be targeted, while avoiding the more traditional gene activation-enhancing responses that dominate estrogen-induced epithelial proliferation. This remains a primary goal of efforts to dissect vascular cell ER biology.