Sequential segments of rat aorta incubated in vitro exhibit a characteristic activity pattern for the metabolism of L-arginine, the substrate for nitric oxide synthase, and for the content of guanosine 3',5'-cyclic monophosphate (cGMP), the mediator of nitric oxide relaxation of vascular smooth muscle. Highest values were observed just distal to the arch and diminish peripherally. Prior removal of the endothelium, treatment with ouabain, or replacement of ambient medium Na+ decreased L-arginine uptake and metabolism and eliminated the pattern of regional differentiation. Removal of endothelium reduced the cGMP content with loss of the regional pattern. A favorable extracellular/intracellular Na+ gradient is required for the moiety of L-arginine uptake destined for metabolism in the endothelial cell. Replacement of ambient Na+ or treatment with ouabain also decreases markedly the L-arginine metabolism and uptake in cultured rat aortic endothelial cells. When aortic segments were tested with five additional substances, L-leucine uptake alone followed a regional pattern similar to that for L-arginine, and no such pattern was observed for the uptake of L-alanine, alpha-aminoisobutyrate, 3-methylglucose, or Ca2+.