Sex steroids exert profound and controversial effects on cardiovascular function. For example, estrogens have been reported to either ameliorate or exacerbate coronary heart disease. Although estrogen dilates coronary arteries from a variety of species, the molecular basis for this acute, non-genomic effect is unclear. Moreover, we know very little of how estrogen affects human coronary artery smooth muscle cells (HCASMC). The purpose of this study was to elucidate non-genomic estrogen signal transduction in HCASMC. We have employed tissue (arterial tension studies), cellular (single-channel patch-clamp, fluorescence), and molecular (protein expression) techniques to now identify novel targets of estrogen action in HCASMC: Type 1 (neuronal) NOS and phosphatidyl inositol (PI) 3 kinase / Akt. 17-estradiol (E2) increased NO-stimulated fluorescence in HCASMC, and cell-attached patch-clamp experiments revealed that stimulation of nNOS leads to increased activity of calcium-activated potassium (BK_Ca) channels in these cells. Furthermore, over-expression of nNOS protein in HCASMC greatly enhanced BK_Ca channel activity. Immunoblot studies demonstrated that E2 enhances Akt phorphorylation in HCASMC, and that wortmannin, an inhibitor of PI3 kinase, attenuated E2-stimulated channel activity, NO production, Akt phosphorylation, and estrogen-stimulated coronary relaxation. These studies implicate the PI3 kinase/Akt signaling axis as an estrogen transduction component in vascular smooth muscle cells. We conclude, therefore, that estrogen opens BK_Ca channels in HCASMC by stimulating nNOS via a transduction sequence involving PI3 kinase and Akt. These findings now provide a molecular mechanism that can explain the clinical observation that estrogen enhances coronary blood flow in patients with diseased or damaged coronary arteries.