BACKGROUND Hydrogen peroxide (H2O2) plays a key role in the control of resistance artery (RA) tone and is hypothesized as an endothelial-derived hyperpolarizing factor. METHODS In this study, we investigated the effects of the same concentration of exogenous H2O2 on mouse mesenteric RA tone induced by intraluminal pressure, G-protein coupled receptor activation and KCl. RAs were cannulated in an arteriograph in the absence or presence of vasoconstrictors. RESULTS RA developed myogenic tone (MT) in response to stepwise pressure increases. Under phenylephrine, H2O2 induced a dose-dependent (1-50 micromol/l) vasodilation with maximum dilation at 50 micromol/l. H2O2 at 50 micromol/l induced a full dilation of RA under MT or contraction by phenylephrine that was independent of nitric oxide synthase, cyclooxygenase, endothelium and potassium channels iberiotoxin and apamin sensitive. The Ca2+ channel inhibitor, nimodipine, significantly blocked MT and also the contraction to phenylephrine and KCl. Under these conditions, H2O2 had no effect on RA diameter. Under KCl, the same concentration of H2O2 induced a potent vasoconstriction. This contraction involved p38 mitogen-activated protein-kinase activation but not ERK1/2. CONCLUSIONS These findings provide the first evidence showing that the same and low concentrations of H2O2 can act as a relaxing factor but also as a vasoconstrictor under conditions in which hyperpolarization is compromised.