Previously we demonstrated that 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) produce reductions in myocardial infarct size. Although it is assumed that this effect of the EETs is due to a specific interaction with a membrane bound receptor, no data suggest that novel EET antagonists selectively block EET actions in dogs. Our goals were to investigate the effects of 11,12- and 14,15-EET, the EPHX2 inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), and the selective EET antagonist, 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) on infarct size in barbital anesthetized dogs. Furthermore, the effect of 14,15-EEZE on the cardioprotective actions of the mitochondrial K_ATP channel opener, diazoxide, was investigated. Both 11,12- and 14,15-EET reduced infarct size (expressed as a percent of the area at risk, IS/AAR) from 21.8 ± 1.6% to 8.7 ± 2.2% and 9.4 ± 1.3%, respectively. Similarly, AUDA significantly reduced IS/AAR from 21.8 ± 1.6% to 14.4 ± 1.2% (low dose) and 9.4 ± 1.8% (high dose), respectively. The combination of low dose AUDA with 14,15-EET further reduced IS/AAR to 5.8 ± 1.6% (P< 0.05). Diazoxide also reduced IS/AAR (10.2 ± 1.9%). In contrast, 14,15-EEZE had no effect on IS/AAR (21.0 ± 3.6%) but abolished the effect of 11,12-EET (17.8 ± 1.4%), 14,15-EET (19.2 ± 2.4%) and AUDA (19.3 ± 1.6%) but not diazoxide (10.4 ± 1.4%). These results suggest that activation of the EET pathway by exogenous EETs or indirectly by blocking EET metabolism produced marked cardioprotection. These data also suggest that 14,15-EEZE is not blocking the mito K_ATP channel.