Endothelial nitric oxide synthase (eNOS) is regulated both by caveolin-1 and 17β-estradiol (E₂). Temporal relationships between effects of estrogen on caveolin-1 and nitric oxide (NO) are not known. Therefore, this study was designed to determine whether estrogen regulates caveolin-1 and, if so, whether such regulation corresponds to changes in nitrite/nitrate (NO_x) production. Bovine aortic endothelial cells (BAECs) were cultured in the absence and presence of 17β-estradiol or 17α-estradiol (10⁻⁸ and 10⁻¹⁰ M) for 12, 24, and 48 h. eNOS protein expression and NO_x production increased significantly after 24 h but not after 12-h treatment with 17β- and not 17α-estradiol. Both mRNA and protein for caveolin-1 were increased significantly only after 48-h treatment with E₂, but eNOS protein and NO_x production were decreased compared with cells treated for 24 h. These increases in caveolin-1 were inhibited by the estrogen receptor antagonist ICI-182,780 (10⁻⁶ M). Results of this study suggest that E₂ stimulates caveolin-1 transcription and translation through estrogen receptor-mediated mechanisms. The results further suggest that estrogen may indirectly regulate NO_x through caveolin-1 expression, which inhibits eNOS catalytic activity.