The ability of estrogens to produce rapid changes in cellular function has been firmly established. The question remains whether these changes are mediated by a modified form of the nuclear estrogen receptor (ER) that is associated with the plasma membrane (mER) or by a completely novel membrane receptor. Therefore, we characterized the biochemical properties of the nuclear and membrane-associated ERs expressed endogenously in a rat hypothalamic endothelial cell line (D12). Radioligand binding experiments using D12 membrane fractions showed that these cells exhibit properties consistent with a binding site specific for estrogens (mER). Equilibrium binding assays using [125I]16-alpha-iodo-3,17- beta-estradiol revealed saturable binding to mER, an affinity value similar to nuclear ER, with differing receptor expression levels. Competition assays revealed that 9 of 12 ER ligands tested had comparable affinities for mER and ER. For example, 17-alpha-estradiol and estrone had similar binding characteristics for both receptors while differences were noted for raloxifene, 17beta-estradiol (E2), and genistein. Western blot and immunocytochemical analyses using antibodies specific for ERalpha confirmed that D12 cells expressed a membrane-associated protein with a molecular mass (67 kDa) similar to that of ERalpha that colocalized with caveolae-enriched membranes. A rapid increase in intracellar Ca2+ levels in the presence of E2 suggests that mER can mediate physiologic changes through calcium mobilization. These data support the expression of mER in these brain-derived endothelial cells that is similar to, but biochemically distinguishable from, nuclear ERalpha.