Cytochrome P450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce improvements in the recovery of left ventricular developed pressure in isolated mouse hearts following ischemia/reperfusion injury partially via activation of ATP-sensitive potassium channels (K_ATP), however, the direct effects of the EETs on myocardial infarct size in a large mammalian heart and the role of K_ATP channels in their cardioprotective effects have not been investigated. In the present study, we demonstrated that two major regioisomers of the CYP epoxygenase pathway, 11,12-EET (0.128 mg/kg) and 14,15-EET (0.128 mg/kg) significantly reduced myocardial infarct size in dogs as compared with control (22.1±1.8% (control), whether administered 15 minutes prior to 60 minutes of coronary occlusion [(6.4±1.9%, 11,12-EET), (8.4±2.4%, 14.15-EET)] or 5 minutes prior to 3 hours of reperfusion [(8.8±2.1%, 11,12-EET, (9.7±1.4%, 14,15-EET)]. Pretreatment with the expoxide hydrolase metabolite of 14,15-EET, 14,15-DHET, had no effect on infarct size. The protective effect of pretreatment with 11,12-EET was completely abolished (24.3±4.6%) in the presence of the K_ATP channel antagonist glibenclamide (1 mg/kg, iv). We further demonstrated that one 5-min period of ischemic preconditioning (IPC) reduced infarct size to a similar extent (8.7±2.8%) to that observed with the EETs. Interestingly, the selective CYP epoxygenase inhibitor, MS-PPOH, at a dose which had no effect on IS/AAR alone (21.0±3.5%) did not block the effect of IPC (10.5±2.0%) which suggests that increases of endogenous EETs are not essential for the protective effect of IPC in this model. On the other hand, administration of MS-PPOH concomitantly with DDMS, a selective inhibitor of endogenous CYP -hydroxylases, which is markedly cardioprotective, completely abolished the reduction in IS/AAR produced by DDMS (4.6±1.2%, DDMS; 22.2±3.4%, MS-PPOH+DDMS). These data collectively suggest that several major regioisomers of the CYP epoxygenase pathway, 11,12-EET and 14,15-EET, produce marked reductions in IS/AAR in dogs and that a major portion of their cardioprotective effect occurs at reperfusion, whereas, the endogenous formation of these compounds during IPC is not necessary for the protective effects of IPC. Conversely, inhibition of CYP epoxygenases and endogenous EET formation by MS-PPOH, in the presence of the CYP -hydroxylase inhibitor, DDMS, blocked cardioprotection which suggests that endogenous EETs are important for the beneficial effects observed when CYP -hydroxylases are inhibited. Finally, the protective effects of the EETs are mediated by cardiac KATP channel opening.