Although 17β-estradiol (E2) is known to induce upregulation of endothelial nitric oxide synthase (eNOS) gene expression, the underlying mechanism is largely unclear. In this study, we show that 17β-estradiol (E2) increases eNOS expression through the binding of estrogen receptor α (ERα) to its promoter. E2 (1 nM) increased both eNOS and nitric oxide production, peaking at 24 h, in bovine aortic endothelial cells, and these increases were accompanied by increment of ERα. To map the estrogen-responsive elements of the eNOS gene for ERα binding under E2, we used three constructs, which were an eNOS gene promoter (−1,600 to +22 nucleotides) fused with a luciferase reporter gene (pGL2-eNOS(−1,600)). However, progressive 5′-deletion from −1,600 to −429 of promoter was attenuated the eNOS transcription. Electrophoretic mobility shift and anti-ERα antibody supershift analyses showed that ERα bound to the estrogen-responsive element (ERE) site, located at −756 to −748 of the eNOS promoter. Our data demonstrate that the interaction between ERα and the estrogenresponsive elements within the eNOS promoter may represent a mechanism for E2-induced gene expression.