Background-We recently reported that arterial superoxide (O 2Ϫ ) is augmented by increased endothelin-1 (ET-1) in deoxycorticosterone acetate (DOCA)-salt hypertension, a model of low renin hypertension. Tetrahydrobiopterin (BH 4 ), a potent reducing molecule with antioxidant properties and an essential cofactor for endothelial nitric oxide synthase, protects against O 2 Ϫ -induced vascular dysfunction. However, the interaction between O 2 Ϫ and BH 4 on endothelial function and the underlying mechanisms are unknown. Methods and Results-The present study tested the hypothesis that BH 4 deficiency due to ET-1-induced O 2 Ϫ leads to impaired endothelium-dependent relaxation and that gene transfer of human guanosine 5Ј-triphosphate (GTP) cyclohydrolase I (GTPCH I), the first and rate-limiting enzyme for BH 4 biosynthesis, reverses such deficiency and endothelial dysfunction in carotid arteries of DOCA-salt rats. There were significantly increased arterial O 2 Ϫ levels and decreased GTPCH I activity and BH 4 levels in DOCA-salt compared with sham rats. Treatment of arteries of DOCA-salt rats with the selective ET A receptor antagonist ABT-627, NADPH oxidase inhibitor apocynin, or superoxide dismutase (SOD) mimetic tempol abolished O 2 Ϫ and restored BH 4 levels. Basal arterial NO release and endothelium-dependent relaxations were impaired in DOCA-salt rats, conditions that were improved by apocynin or tempol treatment. Gene transfer of GTPCH I restored arterial GTPCH I activity and BH 4 levels, resulting in reduced O 2 Ϫ and improved endothelium-dependent relaxation and basal NO release in DOCA-salt rats. Conclusions-These