Flow-induced dilation (FID) is dependent largely on hyperpolarization of vascular smooth muscle cells (VSMCs) in human coronary arterioles (HCA) from patients with coronary disease. Animal studies show that shear stress induces endothelial generation of hydrogen peroxide (H₂O₂), which is proposed as an endothelium-derived hyperpolarizing factor (EDHF). We tested the hypothesis that H₂O₂ contributes to FID in HCA. Arterioles (135±7 μm, n=71) were dissected from human right atrial appendages at the time of cardiac surgery and cannulated with glass micropipettes. Changes in internal diameter and membrane potential of VSMCs to shear stress, H₂O₂, or to papaverine were recorded with videomicroscopy. In some vessels, endothelial H₂O₂ generation to shear stress was monitored directly using confocal microscopy with 2′,7′-dichlorofluorescin diacetate (DCFH) or using electron microscopy with cerium chloride. Catalase inhibited FID (%max dilation; 66±8 versus 25±7%; P<0.05, n=6), whereas dilation to papaverine was unchanged. Shear stress immediately increased DCFH fluorescence in the endothelial cell layer, whereas treatment with catalase abolished the increase in fluorescence. Electron microscopy with cerium chloride revealed shear stress–induced increase in cerium deposition in intimal area surrounding endothelial cells. Exogenous H₂O₂ dilated (%max dilation; 97±1%, ED₅₀; 3.0±0.7×10⁻⁵ mol/L) and hyperpolarized HCA. Dilation to H₂O₂ was reduced by catalase, 40 mmol/L KCl, or charybdotoxin plus apamin, whereas endothelial denudation, deferoxamine, 1H-^1,2,4-oxadiazole-[4,3-a]quinoxalin-1-one, or glibenclamide had no effect. These data provide evidence that shear stress induces endothelial release of H₂O₂ and are consistent with the idea that H₂O₂ is an EDHF that contributes to FID in HCA from patients with heart disease. The full text of this article is available at http://www.circresaha.org.