ERα and ERβ serve classically as transcription factors, and ERα also mediates nongenomic responses to E2 such as the activation of endothelial nitric oxide synthase (eNOS). In contrast, the nongenomic capacities of endogenous ERβ are poorly understood. We evaluated eNOS activation by E2 in cultured endothelial cells that express endogenous ERβ to determine whether the ERβ isoform has nongenomic action and to reveal the subcellular locale of that function. A subpopulation of ERβ was localized to the endothelial cell plasma membrane, overexpression of ERβ enhanced rapid eNOS stimulation by E2, and the response to endogenous ER activation was inhibited by the ERβ-selective antagonist RR-tetrahydrochrysene (THC). eNOS activation through ERβ was reconstituted and shown to occur independent of ERα in COS-7 cells, and ERβ protein in COS-7 was directed to the plasma membrane. THC also blunted E2 activation of eNOS in isolated endothelial cell plasma membranes. Furthermore, ERβ protein was detected and THC attenuated E2 stimulation of eNOS in isolated endothelial cell caveolae, and functional ERβ-eNOS coupling was recapitulated in caveolae from transfected COS-7 cells. These findings in the ER-eNOS signaling paradigm indicate that endogenous ERβ has nongenomic action in caveolae.