Cardiac ischemia and reperfusion are associated with loss in the activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH). Pharmacological stimulation of PDH activity improves recovery in contractile function during reperfusion. Signaling mechanisms that control inhibition and reactivation of PDH during reperfusion were therefore investigated. Using an isolated rat heart model, we observed ischemia-induced PDH inhibition with only partial recovery evident on reperfusion. Translocation of the redox-sensitive δ-isoform of protein kinase C (PKC) to the mitochondria occurred during reperfusion. Inhibition of this process resulted in full recovery of PDH activity. Infusion of the δPKC activator H₂O₂ during normoxic perfusion, to mimic one aspect of cardiac reperfusion, resulted in loss in PDH activity that was largely attributable to translocation of δPKC to the mitochondria. Evidence indicates that reperfusion-induced translocation of δPKC is associated with phosphorylation of the αE1 subunit of PDH. A potential mechanism is provided by in vitro data demonstrating that δPKC specifically interacts with and phosphorylates pyruvate dehydrogenase kinase (PDK)2. Importantly, this results in activation of PDK2, an enzyme capable of phosphorylating and inhibiting PDH. Thus, translocation of δPKC to the mitochondria during reperfusion likely results in activation of PDK2 and phosphorylation-dependent inhibition of PDH.