Previous studies have indicated that acute increases in shear stress can stimulate endothelial nitric oxide synthase (eNOS) activity through increased PI3 kinase/Akt signaling and phosphorylation of serine 1177 (ser1177). However, the mechanism by which shear stress activates this pathway has not been adequately resolved nor has the potential role of reactive oxygen species (ROS) been evaluated. Thus, the purpose of this study was to determine if shear mediated increases in ROS play a role in stimulating ser1177 phosphorylation and NO signaling in pulmonary arterial endothelial cells (PAEC) exposed to acute increases in shear stress. Our initial studies demonstrated that although shear stress did not increase superoxide levels in PAEC there was an increase in H₂O₂ levels. The increases in H₂O₂ were associated with a decrease in catalase activity but not protein levels. In addition, we found that acute shear stress caused an increase in eNOS phosphorylation at ser1177 phosphorylation and a decrease in phosphorylation at Threonine 495 (Thr495). We also found that the over-expression of catalase significantly attenuated the shear mediated increases in H₂O₂, phospho-ser1177 eNOS, and NO generation. Further investigation identified a decrease in PKC activity in response to shear stress and the over-expression of PKC attenuated the shear mediated decrease in Thr495 phosphorylation, the increase in NO generation, and this led to increased eNOS uncoupling. PKC over-expression also attenuated ser1177 phosphorylation through a post-translational increase in catalase activity, mediated via a serine phosphorylation event, reducing shear mediated increases in H₂O₂. Together our data indicate that shear stress decreases PKC activity, altering the phosphorylation pattern catalase, leading to decreased catalase activity and increased H₂O₂ signaling and this in turn leads to increases in phosphorylation of eNOS at ser1177 and NO generation.