Experiments were designed to test the hypothesis that elevated levels of endothelin 1 (ET-1) in the vasculature activate NADPH oxidase and/or uncoupled nitric-oxide synthase (NOS), resulting in O2-. production, and mediate increased constriction. Rat aortic rings were incubated with ET-1 or vehicle in the presence and absence of superoxide dismutase (SOD), ebselen (glutathione peroxidase mimetic), apocynin (NADPH oxidase inhibitor), L-NAME (Nω-nitro-L-arginine methyl ester) (NOS inhibitor), tetrahydrobiopterin (BH4) (NOS cofactor), or selective ETA and ETB receptor antagonists (BQ-123 [cyclo(D-Asp-Pro-D-Val-Leu-D- Trp)] and A-192621 [[2R-(4-propoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N-(2,6- diethylphenyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid]], respectively). O2-. production was monitored by oxidized dihydroethidine staining and/or lucigenin chemiluminescence. ET-1 significantly increased O2-. production compared with vehicle. SOD, ebselen, and apocynin inhibited the ET-1-induced increase in O2-. in intact and endothelium-denuded aorta. L-NAME and BH4 inhibited the ET-1-induced increase in O2-. in intact tissue, whereas these two compounds had no effect on ET-1-induced O 2-. in endotheliumdenuded aorta. Preincubation with BQ-123 or A-192621, individually, had no effect on ET-1-induced O2-.; however combining both antagonists inhibited the ET-1-stimulated increase in O2-.. Rat aortic rings were incubated with ET-1 or vehicle in the presence or absence of sepiapterin (BH4 synthesis substrate) or apocynin and mounted on wire myographs to determine isometric force generation in response to increasing KCl concentrations. ET-1 increased the contractile response to KCl compared with vehicle. Treatment with either sepiapterin or apocynin attenuated the ET-1-mediated increase with no effect of sepiapterin or apocynin alone. These data support the hypothesis that ET-1 increases vascular tone, in part, through ETA/ETB receptor activation of O2-. production from NADPH oxidase and NOS uncoupling. Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics.