Human immunodeficiency virus (HIV)-infected patients have a higher incidence of oxidative stress, endothelial dysfunction, and cardiovascular disease than uninfected individuals. Recent reports demonstrate that viral proteins up-regulate reactive oxygen species, which may contribute to elevated cardiovascular risk in HIV-1 patients. Here we employ an HIV-1 transgenic rat model to investigate the physiological effects of viral protein expression on the vasculature. Markers of oxidative stress in wild-type and HIV-1 transgenic rats were measured using electron spin resonance, fluorescence microscopy and various molecular techniques. Relaxation studies were completed on isolated aortic rings, and mRNA and protein were collected to measure changes in expression of nitric oxide (NO) and superoxide sources. HIV-1 transgenic rats display significantly less NO-hemoglobin, serum nitrite, serum S-nitrosothiols and aortic tissue NO, and impaired endothelium-dependent vasorelaxation versus wild-type rats. NO reduction is not attributed to differences in endothelial NO synthase (eNOS) protein expression, eNOS-Ser¹¹⁷⁷ phosphorylation or tetrahydrobiopterin availability. Aortas from HIV-1 transgenic rats have higher levels of superoxide and 3-nitrotyrosine, but do not differ in expression of superoxide-generating sources NADPH oxidase or xanthine oxidase. However, transgenic aortas display decreased superoxide dismutase and glutathione. Administering the glutathione precursor procysteine decreases superoxide, restores aortic NO levels and NO-hemoglobin, and improves endothelium-dependent relaxation in HIV-1 transgenic rats. These results show that HIV-1 protein expression decreases NO and causes endothelial dysfunction. Diminished antioxidant capacity increases vascular superoxide levels, which reduce NO bioavailability and promote peroxynitrite generation. Restoring glutathione levels reverses HIV-1 protein-mediated effects on superoxide, NO, and vasorelaxation.