Reactive oxygen species (ROS) and endothelin-1 (ET-1) contribute to vascular pathophysiology in obesity. In this context, whether ET-1 modulates hydroxyl radical (·OH) formation and the function of ROS/·OH in obesity is not known. In the present study, formation and function of ROS including ·OH were investigated in the aorta of lean and leptin-deficient obese male mice. Hydroxyl radical formation was detected ex vivo using terephthalic acid in intact aortic rings and the involvement of ROS in ET-1-mediated vasoreactivity was analyzed using the antioxidant EPC-K1, a combination of -tocopherol and ascorbic acid. Generation of either ·OH, ·O₂^-, and H₂O₂ was strongly inhibited by EPC-K1 (all P<0.05). In obese mice, basal vascular ·OH formation and ROS activity was reduced by 3-fold and 5-fold, respectively (P<0.05 vs. lean). ET-1 markedly enhanced ·OH formation in lean (6-fold, P<0.05 vs. untreated), but not in obese mice. Obesity increased ET-1-induced contractions (P<0.05 vs. lean) and ROS scavenging further enhanced the response (P<0.05 vs obese control). Exogenous ROS including ·OH caused stronger vasodilation in obese animals (P<0.05 vs. lean) whereas endothelium-dependent relaxation was similar between the lean and obese animals. In conclusion, we present a sensitive method allowing ex vivo measurement of vascular ·OH generation and provide evidence that ET-1 regulates vascular ·OH formation. The data indicate that in obesity vascular formation of ROS including ·OH is lower whereas the sensitivity is increased, suggesting a novel and important role of ROS including ·OH in the regulation of vascular tone in disease status associated with increased body weight.