Estrogen (E) has nitric oxide (NO)-mediated vasodilatory effects in specific vascular beds including the pulmonary circulation. Since fetal E levels rise markedly with parturition, E may play a role in the regulation of NO-mediated pulmonary vasodilation at birth. We therefore tested the hypothesis that E stimulates endothelial NO synthase (eNOS) in fetal pulmonary artery endothelial cells (PAEC). To examine the direct effects of the hormone, NOS activity was assessed in cultured ovine fetal PAEC by measuring3 H-L-arginine conversion to 3H-L-citrulline in intact cells over 15 min. NOS activity in the presence of 10-14 to 10 -6M estradiol-17β (E2β) rose in a dose-dependent manner, with a threshold concentration of 10-10M and a maximal increase of 294% at 10-8M E2β. This effect was also detectable within 5 min of E2β exposure (248% increase), and it was comparable to that obtained with acetylcholine (250% increase) which had a threshold concentration of 10-8M. NOS activity in cell lysates was similar in control and E2β-treated cells, indicating that there is not upregulation of NOS expression. The estrogen receptor (ER) antagonist ICI 182,780 fully inhibited E2β-stimulated NOS activity in the intact cells. In addition, ER mRNA expression was evident in PAEC in reverse transcription-PCR assays, and ER protein expression was demonstrated by immunocytochemistry. E2β-stimulated NOS activity was also fully inhibited by removal of extracellular calcium (Ca++), and by LaCl3 (10-4M) and NiCl2 (10-3M), which are inhibitors of Ca++ influx. In contrast, intracellular Ca++ store depletion with thapsigargin (10-6M) had no effect on E2β-stimulated NOS activity. Furthermore, inactivation of Ca++-dependent potassium channels with tetraethylammonium ions (5× 10-4M) fully inhibited E2β-stimulated NOS activity. Thus, physiologic concentrations of E acutely stimulate eNOS activity in fetal PAEC by a mechanism that involves the activation of endothelial ER and Ca++-dependent potassium channels, leading to Ca++ influx. This mechanism may play a role in NO-mediated pulmonary vasodilation at birth, and it may underly E responsiveness in other vascular beds.