Background and Purpose-Endothelial nitric oxide synthase produces superoxide under physiological conditions leading to hydrogen peroxide (H2O2) -dependent dilations to acetylcholine in isolated mouse cerebral arteries. The purpose of this study was to investigate whether H2O2 was involved in flow-mediated dilation (FMD). Methods-Cerebral arteries were isolated from 12 +/- 2-week-old C57B1/6 male mice. FMD (0 to 10 mu L/min, 2-mu L step increase at constant internal pressure) was induced in vessels preconstricted with phenylephrine (30 mu mol/L). Simultaneously to diameter acquisition, H2O2 or nitric oxide production was detected by the fluorescent dyes CMH(2)CFDA or 4,5-diaminofluorescein diacetate, respectively. Results are expressed as mean +/- SEM of 6 to 8 mice. Results-FMD (at 10 mu L/min, 25 +/- 3% of maximal diameter) was prevented (P<0.05) by endothelium removal (6 +/- 1%) or endothelial nitric oxide synthase inhibition with N-nitro-L-arginine (11 +/- 1%) but not by the specific nitric oxide scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl3-oxide (24 +/- 3%). Addition of PEG-catalase and silver diethyl dithio-carbamate (superoxide dismutase inhibitor) reduced (P<0.05) FMD to 10 +/- 2% and 15 +/- 1%, respectively. Simultaneously to FMD, H2O2-associated rise in fluorescence (+133 +/- 19 a.u.) was prevented by N-nitro-L-arginine, PEG-catalase, and silver diethyl dithio-carbamate (+55 +/- 10, +64 +/- 4, and +50 +/- 10 a.u., respectively; P<0.05). Inhibition of FMD by PEG-catalase was fully restored by the addition of tetrahydrobiopterin, a cofactor of endothelial nitric oxide synthase (23 +/- 3%); this functional reversal in dilation was associated with the simultaneous increase in nitric oxide-associated fluorescence (+418 +/- 58 a.u., P<0.05), which was prevented by 2-phenyl-4,4,5,5-tetramethylimidazoline1-oxyl3-oxide (+93 +/- 26 a.u.). Akt inhibition with triciribine prevented FMD and H2O2-associated rise in fluorescence (3 +/- 1% and +23 +/- 4% a.u., respectively; P<0.05), but not acetylcholine-induced dilation. Conclusion-In healthy C57B1/6 mouse cerebral arteries, Akt-dependent activation of endothelial nitric oxide synthase-derived H2O2 mediates flow-dependent dilation. (Stroke. 2009;40:1827-1833.)