Experiments were designed to determine whether nitric oxide was the mediator of increased endothelium-dependent relaxations in veins proximal to an arteriovenous fistula. A fistula was prepared between femoral arteries and veins in dogs. After 6 wk, veins proximal to the fistula were removed, cut into rings, and suspended for the measurement of isometric force in organ chambers. In some rings the endothelium was removed deliberately. NG-monomethyl-L-arginine (L-NMMA) caused contraction in three of six fistula-operated veins with and without endothelium. In rings contracted submaximally with prostaglandin F2 alpha, acetylcholine and the alpha 2-adrenergic agonist UK-14,304 cause e tylcholine and the alpha 2-adrenergic agonist UK-14,304 caused endothelium-dependent, concentration-dependent relaxations that were greater in fistula compared with sham-operated veins. These relaxations were reduced by L-NMMA. Calcium ionophore A23187 caused comparable endothelium-dependent relaxations in fistula- and sham-operated veins that were unaffected by L-NMMA. There were no differences in either calcium-dependent or -independent activity of nitric oxide synthase isolated from fistula- and sham-operated veins. Positive staining for nitric oxide synthase was present in both the endothelium and media of fistula-operated veins. These results indicate that nitric oxide mediates increased endothelium-dependent relaxations to acetylcholine and alpha 2-adrenergic agonists in fistula-operated veins. Therefore, chronic increases in blood flow and oxygen tension modify selectively receptor-coupled production of nitric oxide in endothelium and smooth muscle of veins.