In hypertension, the production of reactive oxygen species (ROS) induced by contractile agonists is increased in blood vessels, mainly in endothelial cells (EC). This study aimed to evaluate the role of the endothelium and ROs to the contraction induced by phenylephrine (PE) in isolated aorta from renal hypertensive rat (2K-1C) and normotensive rat (2K). Concentration-effect curves for PE were constructed in intact endothelium (E+) and in denuded rat aorta (E-), in the absence or in the presence of the superoxide scavenger Tiron or Catalase. Expression of Ser1177 phosphorylated eNOS was evaluated by Western-Blotting. In isolated EC, it was measured the fluorescence intensity (FI) of the ROS sensitive dye (DHE) by flow citometry. The EC were stimulated with PE and the IF was measured in the absence (control) and presence of Tiron, or the enzyme that catalyses the degradation of H2O2, Catalase 300 and 3000U, or the combination of both. Aortas E+/E- were stimulated or not (basal) with PE and H2O2 production was measured. The contraction to PE was lower in aortas E+ than in aortas E-, in 2K and 2K-1C. In aortas E+, the Emax was decreased only in 2K-1C. Catalase partially reversed the decreased contraction to PE in 2K-1C E+. PE induces higher Ser1177 phosphorylation of eNOS in 2K-1C aorta than in 2K. The basal IF to DHE was not different between the EC from 2K and 2K-1C. PE increased the IF only in EC from 2K-1C. The increase in EROs was reduced to the basal levels by the antioxidants. In 2K and 2K-1C, the basal H2O2 production was higher in aortas E+ than E-. However, PE stimulated higher H2O2 production in 2K-1C (E+ /E-) as compared with 2K (E+/E-). In conclusion, The reduced contraction to PE in E+ rat aorta from 2K-1C is due to high eNOS-Ser1177 phosphorylation and H2O2 production in the endothelial cells. Supported by CNPq and Fapesp.