Hydrogen peroxide (H 2 O 2) is produced in vessels during ischemia/reperfusion and during inflammation, both leading to vascular dysfunction. We investigated cellular pathways involved in endothelial nitric oxide synthase (eNOS) phosphorylation at Threonine 495 (Thr 495) in human umbilical vein endothelial cells (HUVECs) exposed to H 2 O 2. HUVECs were exposed to 400 μ M H 2 O 2 for 30 min. Phosphorylation at Thr 495 was assessed by Western blotting and reactive oxygen species (ROS) monitored by flow cytometry. Protein kinase C (PKC) pathways were investigated by pretreatment with PKC-β inhibitor ruboxistaurin or pan-PKC inhibitor GF109203X. In addition, we investigated ROCK and ERK pathways by MEKK1/2 inhibitor U0126 and ROCK inhibitor Y27632. H 2 O 2 increased eNOS phosphorylation at Thr 495 (to 176% vs. control (100%), p< 0.001) along with increased mitochondrial ROS formation (from 19.7 to 45.3%, p< 0.01). This rise in phosphorylation could be prevented by U0126 and Y27632 in a dose-dependent manner, but did not result in lowered mitochondrial ROS formation. Conversely, addition of the antioxidant N-acetyl-L-cysteine only prevented mitochondrial ROS formation but did not prevent phosphorylation of eNOS Thr 495. H 2 O 2-mediated phosphorylation of eNOS Thr 495 is mediated by ROCK and ERK activity, but not by PKC, and is uncoupled from mitochondrial ROS signaling. Furthermore, ERK inhibition increased mitochondrial ROS formation.