Urocortin (Ucn) protects hearts against ischemia and reperfusion injury whether given prior to ischemia or at reperfusion. Here we investigate the roles of protein kinase C, reactive oxygen species, and the mitochondrial permeability transition pore (MPTP) in mediating these effects. In Langendorff-perfused rat hearts, acute Ucn treatment improved hemodynamic recovery during reperfusion after 30 min global ischemia; this was accompanied by less necrosis (lactate dehydrogenase release) and MPTP opening (mitochondrial entrapment of [³H]-2-deoxyglucose). Ucn pre-treatment protected mitochondria against calcium-induced MPTP opening, but only if the mitochondria had been isolated from hearts after reperfusion. These mitochondria also exhibited less protein carbonylation, suggesting that Ucn decreases levels of oxidative stress. In isolated adult and neonatal rat cardiac myocytes, both acute (60 min) and chronic (16 hr) treatment with Ucn reduced cell death following simulated ischemia and re-oxygenation. This was accompanied by less MPTP opening as measured using tetramethylrhodamine methyl ester. The level of oxidative stress during reperfusion was reduced in cells which had been pre-treated with Ucn suggesting that this is the mechanism by which Ucn desensitizes the MPTP to reperfusion injury. Despite the fact that we could find no evidence that either PKC or PKC translocate to the mitochondria following acute Ucn treatment, inhibition of PKC with chelerythrine eliminated the effect of Ucn on oxidative stress. Our data suggests that acute Ucn treatment protects the heart by inhibiting MPTP opening. However, the mechanism appears to be indirect, involving a PKC-mediated reduction in oxidative stress.