Cytochrome P450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce marked reductions in infarct size in canine myocardium whether given prior to an ischemic insult or at reperfusion similar to that produced in preconditioning (IPC) and postconditioning (POC) protocols. However, no studies have addressed the possibility that EETs serve a beneficial role in IPC or POC. In the present study, we tested the hypothesis that EETs may play a role in these 2 phenomena by preconditioning dog hearts with one 5-min period of total coronary occlusion followed by 10 min of reperfusion prior to 60 min of occlusion and 3 h of reperfusion or by postconditioning with three 30-sec periods of reperfusion interspersed with three 30-sec periods of occlusion. To test for a role of EETs in IPC and POC, the selective EET antagonists 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) and/or its polyethyleneglycol derivative, 14,15-EEZE-PEG, were administered 10 min prior to IPC or 5 min prior to POC. Infarct size was determined by tetrazolium staining and coronary collateral blood flow at 30 min of occlusion and reperfusion flow at 3 h by radioactive microspheres. Both IPC and POC produced nearly equivalent reductions in infarct size (IS) expressed as a percent (%) of the area-at-risk (AAR); Control = 21.2±1.2, IPC = 8.3±2.2, POC = 10.1±1.8 (P<0.001). Both 14,15-EEZE and 14,15-EEZE-PEG abolished the cardioprotective effects of IPC and POC at doses which had no effect on IS/AAR when given alone. These results suggest a unique role for endogenous EETs in both IPC and POC.