Ischemic preconditioning confers cardiac protection during subsequent ischemia-reperfusion, in which protein kinase C (PKC) is believed to play an essential role, but controversial data exist concerning the PKC-δ isoform. In an accompanying study , we described metabolic changes in PKC-δ knockout mice. We now wanted to explore their effect on early preconditioning. Both PKC-δ^−/− and PKC-δ^+/+ mice underwent three cycles of 5-min left descending artery occlusion/5-min reperfusion, followed by 30-min occlusion and 2-h reperfusion. Unexpectedly, preconditioning exaggerated ischemia-reperfusion injury in PKC-δ^−/− mice. Whereas ischemic preconditioning increased superoxide anion production in PKC-δ^+/+ hearts, no increase in reactive oxygen species was observed in PKC-δ^−/− hearts. Proteomic analysis of preconditioned PKC-δ^+/+ hearts revealed profound changes in enzymes related to energy metabolism, e.g., NADH dehydrogenase and ATP synthase, with partial fragmentation of these mitochondrial enzymes and of the E₂ component of the pyruvate dehydrogenase complex. Interestingly, fragmentation of mitochondrial enzymes was not observed in PKC-δ^−/− hearts. High-resolution NMR analysis of cardiac metabolites demonstrated a similar rise of phosphocreatine in PKC-δ^+/+ and PKC-δ^−/− hearts, but the preconditioning-induced increase in phosphocholine, alanine, carnitine, and glycine was restricted to PKC-δ^+/+ hearts, whereas lactate concentrations were higher in PKC-δ^−/− hearts. Taken together, our results suggest that reactive oxygen species generated during ischemic preconditioning might alter mitochondrial metabolism by oxidizing key mitochondrial enzymes and that metabolic adaptation to preconditioning is impaired in PKC-δ^−/− hearts.