Reperfusion of ischemic myocardium is essential for tissue salvage but paradoxically contributes to cell death. We hypothesized that activation of potential survival pathways such as p42/p44 MAPK may prevent lethal reperfusion injury. Urocortin is a peptide factor that affects the p42/p44 MAPK signaling pathway. Both isolated and in vivo rat heart models were used to examine the potential for urocortin to prevent reperfusion injury. Isolated rat hearts underwent 35-min regional ischemia and 2-h reperfusion, with urocortin perfused for 20 min from the onset of reperfusion. In the in vivo study, urocortin was administered as an intravenous bolus 3 min before reperfusion with a protocol of 25-min regional ischemia and 2-h reperfusion. Blockade of the p42/p44 MAPK pathway with the inhibitor PD-98059 was used in both models. Urocortin attenuated lethal reperfusion-induced injury both in vitro and in vivo via a p42/p44 MAPK-dependent mechanism. Furthermore, Western blot analysis demonstrated the ability of urocortin to directly upregulate this signaling pathway. In conclusion, we believe that the p42/p44 MAPK-dependent signaling pathway represents an important survival mechanism against reperfusion injury.