ATP dose dependently stimulated the formation and release of nitric oxide (NO) from perfused rabbit aorta. L-Canavanine, an inhibitor of various L-arginine-utilizing enzymes, abolished basal and ATP-induced NO formation and release. ATP increased the accumulation of presumably NO-derived NO2- in the medium of primary cultures of bovine aortic endothelial cells. 15NO, 15NO2- and 15NO3- formation was found when L-[guanido-15N2]arginine was added to the culture medium. We conclude that the terminal guanidino nitrogens of L-arginine are the physiological precursors of endothelium-derived NO.