The relaxation of isolated blood vessels by acetylcholine is dependent on the presence of intact endothelium and its release of a smooth muscle relaxing nitroso-like compound. Pregnancy is associated with altered vascular responsiveness to a variety of agents. Because many of these agents stimulate the release of endothelium-derived relaxing factor, we investigated in vitro the effect of pregnancy on acetylcholine-mediated relaxation of guinea pig uterine and carotid artery rings. The presence of intact and functional endothelium was confirmed both by examination under the scanning electron microscope and by vessel relaxation after the addition of acetylcholine. The addition of acetylcholine to the vessel bath produced dose-dependent relaxation of both carotid and uterine artery segments obtained from pregnant and nonpregnant animals after they had been submaximally preconstricted with phenylephrine. There was a significant increase in both response and efficacy to acetylcholine during pregnancy for both uterine and carotid arteries (p less than 0.0001 for each). The concentrations of endothelial cells (cells per square micrometer) were similar in uterine arteries from pregnant and nonpregnant animals. We conclude that the most likely explanation for these findings is a pregnancy-mediated enhancement of endothelium-derived relaxing factor activity.