We previously observed that H₂O₂ is a coronary metabolic dilator and couples myocardial oxygen consumption to coronary blood flow. Because the chemical actions of H₂O₂ are as an oxidant, and thiol groups are susceptible to oxidation, we hypothesized that coronary metabolic dilation occurs via a redox mechanism involving thiol oxidation. To test this hypothesis we studied the mechanisms of dilation of isolated coronary arterioles to metabolites released by metabolically active (paced at 400 min^-1) isolated cardiac myocytes and compared these responses to H₂O₂. Studies were performed under control conditions and using dithiothreitol (DTT) or N-acetlylcysteine (NAC) to reduce oxidized thiols. Aliquots of conditioned buffer from paced myocytes produced vasodilation of isolated arterioles (peak response 71±6% of maximal dilation); whereas, H₂O₂ produced complete dilation (92±7%). Dilation to either conditioned buffer or to H₂O₂ was significantly reduced by either NAC or DTT. Administration of the fluorochromes monochlorobimane or monobromotrimethylammoniobimane, which covalently label reduced total or extracellular reduced thiols, respectively revealed that H₂O₂ or the conditioned buffer predominately oxidized intracellular thiols. To determine if redox signaling leading to dilation is mediated by the redox sensitive kinase, p38 MAP kinase, we evaluated dilation following administration of the p38 inhibitor SB203580. Inhibition of p38 attenuated dilation to either H₂O₂ or to conditioned buffer, but did not attenuate dilation to nitroprusside. Western analysis for the activated form of p38 (phospho-p38) revealed robust activation of this enzyme by H₂O₂ in isolated aortae. Taken together, our results show that an active component of cardiac metabolic dilation, like that of H₂O₂, produces dilation by oxidation of thiols, which are predominately intracellular, and dependent activation on p38 MAP kinase. Thus coronary metabolic dilation appears to be mediated by redox-dependent signals.