The present study was designed to evaluate the effects of POCA, a carnitine palmitoyltransferase I (CPT I) inhibitor, and pyruvate, a substrate inhibiting fatty acid (FA) oxidation, on post-ischemic cardiac FA accumulation on the one hand, and hemodynamic recovery and loss of cellular integrity on the other. To this end isolated, working rat hearts, receiving glucose (11 mM) as substrate, were subjected to 45 min of no-flow ischemia and 30 min of reperfusion. Hearts were perfused with or without POCA (10 microM) and/or pyruvate (5 mM). In the control group the FA content increased significantly during ischemia and remained elevated during reperfusion. Administration of POCA did not affect functional recovery and LDH release significantly, but resulted in about two-fold increased FA levels upon reperfusion as compared to glucose-perfused hearts. Pyruvate markedly improved functional recovery. Addition of this substrate did not affect lactate dehydrogenase (LDH) release, but enhanced FA accumulation during reperfusion. The combined administration of pyruvate and POCA nullified the positive effect of pyruvate on hemodynamic recovery, aggravated LDH release, and further enhanced the accumulation of FAs. The adenine nucleotide content of reperfused hearts was comparable for all groups investigated. In conclusion, during transient ischemia POCA and pyruvate markedly increased cardiac FA accumulation through inhibition of the oxidation of FAs released from endogenous lipid pools. No clear relation was found between the FA content of reperfused hearts and post-ischemic functional recovery.