The study investigates the hypothesis that endothelium-dependent relaxation of vascular smooth muscle is mediated through the formation of cyclic guanosine monophosphate (cGMP). Relaxation of rat thoracic aorta to acetylcholine, histamine, and Ca++ ionophore A23187 was associated with increased cGMP levels, while cAMP levels remained unchanged. Removal of the endothelium prevented relaxation and the increase in cGMP levels. Eicosatetraenoic acid, an inhibitor of lipoygenase and cyclooxygenase, and quinacrine, an inhibitor of phospholipase, prevented and reversed acetylcholine-induced relaxation and increased cGMP levels, respectively. In contrast, sodium nitroprusside-induced relaxation and increased cGMP levels were independent of the presence of the endothelium, exposure to eicosatetraenoic acid, and quinacrine. The results support the hypothesis that vascular smooth muscle relaxation induced by certain agents is dependent on the presence of the endothelium and is mediated through the formation of cGMP within the smooth muscle.The study investigates the hypothesis that endothelium-dependent relaxation of vascular smooth muscle is mediated through the formation of cyclic guanosine monophosphate (cGMP). Relaxation of rat thoracic aorta to acetylcholine, histamine, and Ca++ ionophore A23187 was associated with increased cGMP levels, while cAMP levels remained unchanged. Removal of the endothelium prevented relaxation and the increase in cGMP levels. Eicosatetraenoic acid, an inhibitor of lipoygenase and cyclooxygenase, and quinacrine, an inhibitor of phospholipase, prevented and reversed acetylcholine-induced relaxation and increased cGMP levels, respectively. In contrast, sodium nitroprusside-induced relaxation and increased cGMP levels were independent of the presence of the endothelium, exposure to eicosatetraenoic acid, and quinacrine. The results support the hypothesis that vascular smooth muscle relaxation induced by certain agents is dependent on the presence of the endothelium and is mediated through the formation of cGMP within the smooth muscle.