Objective: Hydrogen peroxide (H2O2) produced by the vascular endothelium is a signaling molecule regulating vascular tone. We hypothesized that H2O2 derived from eNOS activity could play a physiological role in endothelium-dependent dilation of mouse cerebral arteries. Methods: Simultaneous endothelium-dependent dilation and fluorescence-associated free radical (DCF-DA) or NO (DAF-2) production were recorded in isolated and pressurized (60 mm Hg) cerebral artery of C57Bl/6 male mice. Results: Without synergism, N-nitro-l-arginine (l-NNA) or the H2O2 scavengers catalase, PEG-catalase and pyruvate reduced (P2O2 – but not NO – production, sensitive to either l-NNA or catalase, was detected. In cerebral arteries from C57Bl/6·eNOS−/− mice, catalase had no effect on ACh-induced dilation and no H2O2-associated fluorescence was observed. In C57Bl/6 mice, silver diethyldithiocarbamate (DETC), a superoxide dismutase (SOD) inhibitor, but not the specific NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl3-oxide (PTIO), prevented ACh-induced dilation and H2O2 production suggesting that eNOS-derived superoxide is an intermediate in the production of H2O2. The catalase-sensitive ACh-induced dilation was restored by the eNOS cofactor tetrahydrobiopterin (BH4). This reversal was associated with a NO-associated fluorescence sensitive to PTIO but not to catalase. Soluble guanylate cyclase inhibition with 1H-[1,2,4]-oxadiazole-4,3-aquinoxalin-1-one (ODQ) prevented the dilation induced by ACh and by exogenous H2O2. Lastly, l-NNA, PTIO and ODQ – but not DETC, catalase or pyruvate – increased the pressure-dependent myogenic tone, suggesting that eNOS produces NO at rest, but leads to H2O2 during muscarinic stimulation. Conclusion: H2O2-dependent dilation in mouse cerebral arteries appears to be a physiological eNOS-derived mechanism.