Isometric tension was recorded in isolated rings of aorta, carotid, coronary and mesenteric arteries taken from endothelial nitric oxide synthase knockout mice (eNOS(−/−) mice) and the corresponding wild-type strain (eNOS(+/+) mice). The membrane potential of smooth muscle cells was measured in coronary arteries with intracellular microelectrodes.In the isolated aorta, carotid and coronary arteries from the eNOS(+/+) mice, acetylcholine induced an endothelium-dependent relaxation which was inhibited by Nω-L-nitro-arginine. In contrast, in the mesenteric arteries, the inhibition of the cholinergic relaxation required the combination of Nω-L-nitro-arginine and indomethacin.The isolated aorta, carotid and coronary arteries from the eNOS(−/−) mice did not relax in response to acetylcholine. However, acetylcholine produced an indomethacin-sensitive relaxation in the mesenteric artery from eNOS(−/−) mice.The resting membrane potential of smooth muscle cells from isolated coronary arteries was significantly less negative in the eNOS(−/−) mice (−64.8±1.8 mV, n=20 and −58.4±1.9 mV, n=17, for eNOS(+/+) and eNOS(−/−) mice, respectively). In both strains, acetylcholine, bradykinin and substance P did not induce endothelium-dependent hyperpolarizations whereas cromakalim consistently produced hyperpolarizations (−7.9±1.1 mV, n=8 and −13.8±2.6 mV, n=4, for eNOS(+/+) and eNOS(−/−) mice, respectively).These findings demonstrate that in the blood vessels studied: (1) in the eNOS(+/+) mice, the endothelium-dependent relaxations to acetylcholine involve either NO or the combination of NO plus a product of cyclo-oxygenase but not EDHF; (2) in the eNOS(−/−) mice, NO-dependent responses and EDHF-like responses were not observed. In the mesenteric arteries acetylcholine releases a cyclo-oxygenase derivative.