Inhibition of fatty acid oxidation has been reported to be cardioprotective against myocardial ischemic injury; however, recent studies have questioned whether the cardioprotection associated with putative fatty acid oxidation inhibitors, such as ranolazine and trimetazidine, are due to changes in substrate oxidation. Therefore, the goals of this study were to compare the effects of ranolazine with a new fatty acid oxidation inhibitor, CVT-4325 [(R)-1-(2-methylbenzo[d]thiazol-5-yloxy)-3-(4-((5-(4-(trifluoromethyl)phenyl)-1, 2,4-oxadiazol-3-yl)methyl)-piperazin-1-yl)propan-2-ol], on carbohydrate and fatty acid oxidation and on left ventricular (LV) function in the response to ischemia/reperfusion in rat isolated perfused hearts. Metabolic fluxes were determined in hearts perfused in an isovolumic Langendorff mode using 13C nuclear magnetic resonance isotopomer analysis or in isolated working hearts using [14C]glucose and [3H]palmitate, with and without 10 μM ranolazine or 3 μM CVT-4325. Isovolumic perfused hearts were also subjected to 30 min of low-flow ischemia (0.3 ml/min) and 60 min of reperfusion, and working hearts were subjected to 15 min of zero-flow ischemia and 60 min of reperfusion. Regardless of the experimental protocol, ranolazine had no effect on carbohydrate or fatty acid oxidation, whereas CVT-4325 significantly reduced fatty acid oxidation up to ∼7-fold with a concomitant increase in carbohydrate oxidation. At these same concentrations, although ranolazine significantly improved LV functional recovery following ischemia/reperfusion, CVT-4325 had no significant protective effect. These results demonstrate that at pharmacologically relevant concentrations, ischemic protection by ranolazine was not mediated by inhibition of fatty acid oxidation and conversely that inhibition of fatty acid oxidation with CVT-4325 was not associated with improved LV functional recovery. Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics.