Desensitization of a,-adrenergic receptor-mediated contraction occurs in rat aorta after in vitro exposure to a-adrenergic agonists; we previously showed that a component of the desensitization is endothelial cell dependent. Our primary purpose was to examine possible alterations in either the release or action of endothelium-derived relaxing factor (EDRF) in desensitized blood vessels. Rings of rat aorta were desensitized in vitro by exposure to phenylephrine (PE) for 6 h with impaired subsequent ability of PE to induce smooth muscle contraction. PE also induced heterologous desensitization of serotonin-induced contraction, which was blocked by the α1-adrenergic selective antagonist prazosin. Using a “sandwich” bioassay technique, we noted enhanced release of EDRF from the aortic rings that had been previously exposed to PE as compared with controls. The capacity of PE to activate accumulation of inositol monophosphate was impaired in the desensitized blood vessels, both with and without endothelium. Our results suggest that prolonged exposure to a-adrenergic agonists leads to several adaptations in vascular smooth muscle (VSM), including enhanced release of EDRF. Although impaired action of EDRF has been suggested to play a role in diseases such as diabetes and atherosclerosis, our results indicate that release and action of EDRF may be enhanced with prolonged exposure to a-agonists.