Rapid, non-genomic, effects of 17-estradiol (E2) in endothelial cells are postulated to arise from membrane-associated estrogen receptors (ER), which have not been visualized in vascular tissue. To identify membrane ERs we used multiple site-directed ER or ER antibodies to label en face rat cerebral and coronary arterial endothelia. Western blots revealed a novel 55 kDa ER isoform. Three-dimensional images of cells labeled with these antibodies, and markers for the nucleus and caveolin-1, were acquired with a wide-field microscope, deconvolved, and numerically analyzed. We found ER in the nucleus and the cell periphery, where one-third colocalized with caveolin-1. The receptor location was dependent on the antibody's epitope. Human ovarian surface epithelium produced similar results, but in rat myometrium the distribution was epitope-independent and nuclear. ER distribution was predominately intranuclear and epitope-independent. A small amount of ER colocalized with ER within the nucleus. The results were identical in both arterial preparations and insensitive to E2. We postulate that the different ER conformations at the membrane, in the nucleus and between different cell types allow E2 to trigger cell and location specific signaling cascades.