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