Localized exogenous R-tetrahydrobiopterin (R-BH(4)) corrects the deficit in local heat-induced vasodilation (VD) in hypercholesterolemic (HC) human skin through one of two plausible mechanisms: by serving as an essential cofactor to stabilizing endothelial nitric oxide (NO) synthase (eNOS) or through generalized antioxidant effects. We used the stereoisomer S-BH(4), which has the same antioxidant properties but does not function as an essential NOS cofactor, to elucidate the mechanism by which R-BH(4) restores cutaneous VD in HC humans. Intradermal microdialysis fibers were placed in 20 normocholesterolemic (NC), 13 midrange cholesterolemic (MC), and 18 HC (LDL: 94 ± 3, 124 ± 3 and 179 ± 6 mg/dl, respectively) men and women to perfuse Ringer (control site) and R-BH(4). In 10 NC, 13 MC, and 9 HC subjects (LDL: 94 ± 3, 124 ± 3, 180 ± 10 mg/dl), S-BH(4) was perfused at a third microdialysis site. Skin blood flow was measured during a standardized local heating protocol to elicit eNOS-dependent VD. After cutaneous vascular conductance (CVC = LDF/MAP) plateaued, NO-dependent VD was quantified by perfusing N(G)-nitro-l-arginine methyl ester (l-NAME). Data were normalized as %CVC(max). Fully expressed VD (NC: 97.9 ± 2.3 vs. MC: 85.4 ± 5.4, HC: 79.9 ± 4.2%CVC(max)) and the NO-dependent portion (NC: 62.1 ± 3 vs. MC: 45.8 ± 3.9, HC: 35.7 ± 2.8%CVC(max)) were reduced in HC (both P < 0.01 vs. NC), but only the fully expressed VD was reduced in MC (P < 0.01 vs. NC). R-BH(4) increased the fully expressed (93.9 ± 3.4%CVC(max); P < 0.01) and NO-dependent VD (52.1 ± 5.1%CVC(max); P < 0.01) in HC but not in NC or MC. S-BH(4) increased full-expressed VD in HC (P < 0.01) but did not affect NO-dependent VD in HC or MC. In contrast S-BH(4) attenuated NO-dependent VD in NC (control: 62.1 ± 3 vs. S-BH(4): 41.6 ± 7%CVC(max); P < 0.001). Exogenous R-BH(4) restores NO-dependent VD in HC human skin predominantly through NOS coupling mechanisms but increases full expression of the local heating response through generalized antioxidant properties.