['Mitochondrial-derived hydrogen peroxide (H2O2) regulates flow-induced dilation (FID) in microvessels from patients with coronary artery disease. The relationship between ceramide, an independent risk factor for coronary artery disease and a known inducer of mitochondrial reactive oxygen species, and FID is unknown.', 'We examined the hypothesis that exogenous ceramide induces a switch in the mediator of FID from nitric oxide to H2O2.', 'Internal diameter changes of resistance arterioles from human adipose and atrial tissue were measured by video microscopy. Mitochondrial H2O2 production was assayed in arterioles using mito peroxy yellow 1. Polyethylene glycol-catalase, rotenone, and Mito-TEMPO impaired FID in healthy adipose arterioles pretreated with ceramide, whereas N(ω)-nitro-l-arginine methyl ester had no effect. Mitochondrial H2O2 production was induced in response to flow in healthy adipose vessels pretreated with ceramide, and this was abolished in the presence of polyethylene glycol-catalase. Immunohistochemistry demonstrated ceramide accumulation in arterioles from both healthy patients and patients with coronary artery disease. N(ω)-nitro-l-arginine methyl ester reduced vasodilation to flow in adipose as well as atrial vessels from patients with coronary artery disease incubated with GW4869, a neutral sphingomyelinase inhibitor, whereas polyethylene glycol-catalase had no effect.'] Does ceramide change the mediator of flow-induced vasodilation from nitric oxide to hydrogen peroxide in the human microcirculation?