Experiments were designed to analyze the difference in endothelium-dependent responsiveness to acetylcholine between arteries and veins. The effect of endothelium-derived relaxing factor(s) released from femoral arteries of the dog was compared on the coronary artery of the dog, the aorta of the rat, and portal-mesenteric veins of both species. Endothelium-derived relaxing factor(s) released by canine femoral arteries induced comparable relaxation of the canine coronary artery and the aorta of the rat. However, neither the canine nor the rat portal vein relaxed when exposed to endothelium-derived relaxing factors) released by the femoral arterial segments. Endothelium-derived relaxing factor(s) did not affect the action potentials and the spontaneous activity of the rat portal vein. Sodium nitroprusside induced complete relaxation of the canine coronary artery but failed to abolish the spontaneously evoked contractions of the portal veins. These experiments suggest that the longitudinal smooth muscle of portal veins is insensitive to endothelium-derived relaxing factor(s), presumably because of a different sensitivity of guanylate cyclase. Endothelium-derived relaxing factor does not possess calcium-entry blocking properties.