Adenosine A₁ receptor delayed preconditioning (PC) against myocardial infarction has been well described. However there have been limited investigations of the signaling mechanisms mediating this phenomenon. In addition there are multiple conflicting reports on the role of inducible nitric oxide synthase (iNOS) in mediating A₁ late phase PC. The purpose of this study was to determine the roles of the p38 and extracellular signal-regulated kinase (ERK) mitogen activated protein kinases (MAPKs)in in vivo delayed A₁ receptor PC and whether this protection at the myocyte level was due to upregulation of iNOS. Myocardial infarct size was measured in open-chest anesthetized rats 24 hours after treatment with vehicle or the adenosine A1 agonist CCPA (100 µg/kg, i.p.). Additional rats receiving CCPA were pretreated with the p38 inhibitor SB203580 (1 mg/kg, i.p.) or the MEK inhibitor PD098059 (0.5 mg/kg, i.p.). 24 hours after CCPA administration a group of animals was given the iNOS inhibitor 1400W 10 minutes prior to ischemia. Treatment with CCPA reduced infarct size from 48 ± 2% of the area at risk to 28 ± 2%, an effect that was blocked by both SB203580 and PD098059, but not 1400W. Ventricular myocytes isolated 24 hours after CCPA injection exhibited significantly reduced oxidative stress during H₂2O₂ exposure compared to myocytes from vehicle injected animals, an effect that was not blocked by the iNOS inhibitor 1400W. Western blot analysis of whole heart and cardiac myocyte protein samples revealed no expression of iNOS 6 or 24 hours after CCPA treatment. These results indicate that adenosine A₁ receptor delayed PC in the rat are mediated by MAPK-dependent mechanisms, but this phenomenon is not associated with the early or late expression of iNOS.