The present study tested the hypothesis that the pregnancy-associated increase in endothelium-dependent relaxation of the uterine artery was mediated primarily by an increase in nitric oxide (NO) release, resulting in a reduction in smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)). Uterine arteries obtained from nonpregnant and near-term (140 days gestation) pregnant sheep were used. The Ca(2+) ionophore A23187 induced endothelium-dependent relaxations in both nonpregnant and pregnant uterine arteries, with an increased relaxation in the pregnant tissue. In contrast, endothelium-independent relaxations induced by sodium nitroprusside were the same in nonpregnant and pregnant arteries. In addition, removal of the endothelium significantly increased noradrenaline-induced contractions in pregnant, but not nonpregnant, uterine arteries. In accordance, pregnancy increased both basal and A23187-stimulated NO releases in the uterine artery. Simultaneous measurement of tension and [Ca(2+)](i) in the smooth muscle demonstrated a linear correlation with the slope of unity between A23187-induced relaxation and the reduction of [Ca(2+)](i) in both nonpregnant and pregnant uterine arteries. The A23187-induced reduction of [Ca(2+)](i) was significantly enhanced in pregnant, compared with nonpregnant, uterine arteries. The results indicate that pregnancy increases NO release, which, through decreasing [Ca(2+)](i) in the smooth muscle, accounts for the increased endothelium-dependent relaxation of the uterine artery. Signal transduction pathways distal to NO production are not changed by pregnancy.