This study tested the hypothesis that dietary arginine supplementation increases endothelial tetrahydrobiopterin (BH(4)) availability for nitric oxide (NO) synthesis in diabetic rats. Streptozotocin-induced diabetic rats either were given unrestricted access to a casein-based diet (Expt. 1) or were pair-fed the diet on the basis of the food intake per kg of body weight of nondiabetic rats (Expt. 2). Beginning 1 d after vehicle or streptozotocin injection, arginine-HCl (1.51%) or alanine (isonitrogenous control, 2.55%) was added daily to the drinking water for nondiabetic rats, whereas concentrations were adjusted (0.43% arginine-HCl and 0.73% alanine) in the drinking water for diabetic rats (which consumed more water) to ensure isonitrogenous provision. At 2 wk after the initiation of arginine supplementation, coronary endothelial cells and plasma were obtained for the measurement of NO synthesis and metabolites. In both experiments, plasma and endothelial concentrations of N(G)-monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine increased, but those of arginine as well as endothelial BH(4) availability and NO synthesis decreased in diabetic rats, compared with nondiabetic rats. In both diabetic and nondiabetic rats, arginine supplementation increased plasma concentrations of arginine and insulin, endothelial concentrations of arginine and BH(4), and endothelial NO synthesis, but did not affect plasma and endothelial concentrations of methylarginines or plasma concentrations of homocysteine. Dietary arginine supplementation or provision of a BH(4) precursor normalized endothelial NO synthesis in diabetic rats. Arginine supplementation did not affect plasma glucose levels in nondiabetic rats, but reduced body weight loss and plasma glucose levels in diabetic rats. Thus, dietary L-arginine supplementation stimulates endothelial NO synthesis by increasing BH(4) provision, which is beneficial for vascular function and glucose homeostasis in diabetic subjects.