Endothelium-derived relaxing factors (EDRFs) have been previously shown to exert an inhibitory influence on the contractile effects of alpha-adrenoceptor agonists in vascular smooth muscle. alpha 2-Adrenoceptor agonists such as clonidine have been reported to be particularly susceptible to this effect, and it has been suggested that clonidine acts on alpha 2 receptors on endothelial cells to stimulate the release of EDRF. EDRF release is known to be accompanied by increased levels of cGMP in many blood vessels, and it is suggested that cGMP exerts an inhibitory influence on the smooth muscle cells, which tends to counteract the contractile effect of the clonidine. This hypothesis was tested in isolated rings of rat aorta and mesenteric artery using the cGMP lowering agent, 6-anilino-5,8-quinolinedione (LY83583). LY83583 markedly decreased resting levels of cGMP in these vascular preparations and completely prevented both the relaxation and the cGMP elevation normally caused by acetylcholine in rat aorta with intact endothelium. These effects of LY83583 are identical to those observed after mechanical disruption of the endothelium. LY83583 also enhanced the contractile responses to norepinephrine and particularly to clonidine in both aorta and mesenteric artery. The effects of LY83583 on contractile responses to both alpha-adrenoceptor agonists were reversed by low concentrations of 8-bromo-cGMP. Clonidine did not increase cGMP levels in vascular preparations with intact endothelia, in the presence or absence of LY83583. Thus, enhanced release of EDRF by clonidine did not appear to be responsible for the inhibition of its contractile effects observed in the presence of intact endothelial cells. Our results suggest instead that this endothelium-dependent inhibition is due to spontaneous release of EDRF, which results in tonic elevation of cGMP in the vascular smooth muscle. This tonic elevation of cGMP exerts a more marked inhibitory effect against contractions induced by the partial agonist, clonidine, than it does against contractions induced by a full agonist, norepinephrine.