Deficiency of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide (NO) synthase, decreases NO production and increases reactive oxygen species. The purpose of this study was to elucidate the effects of aging on endothelial function and to determine whether the degree of BH4 deficiency is related to aging and oxidative stress. We evaluated forearm blood flow (FBF) responses to acetylcholine (ACh), an endothelium-dependent vasodilator, and isosorbide dinitrate (ISDN), an endothelium-independent vasodilator, before and after co-infusion of BH4 (500 mg/min) in 37 healthy men (mean age, 41+/-18 yr; range, 19-81 yr). FBF was measured using strain-gauge plethysmograph. Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and serum malondialdehyde-modified low-density lipoprotein (MDA-LDL) were measured as indices of oxidative stress. Both ACh and ISDN increased the FBF in a dose-dependent manner in all subjects. Co-infusion of BH4 resulted in a significant increase in ACh-induced vasodilation (from 22.3+/-6.7 to 30.1+/-7.5 mL/min/100 mL tissue, P<0.05). Aging was found to be significantly correlated with ACh-induced vasodilation (r=-0.47, P=0.006), urinary 8-OHdG (r=0.38, P=0.02), serum MDA-LDL (r=0.36, P=0.02), and the change in ACh-induced vasodilation after co-infusion of BH4 (r=0.45, P=0.007). The FBF response to ISDN did not correlate with any parameters. Infusion of N(G)-monomethyl-L-arginine, an NO synthase inhibitor, abolished the BH4-induced enhancement of forearm vasorelaxation evoked by ACh. The increase in FBF after ISDN was not altered by BH4. These findings suggest that a deficiency of BH4 may be involved in the pathogenesis of disturbances in endothelium-dependent vasodilation related to aging through decrease in NO production and increase in oxidative stress.