Activation of the δ-isoform of protein kinase C (δPKC) by certain conditions of oxidative stress results in translocation of the kinase to the mitochondria leading to release of cytochrome c and the induction of apoptosis. In the current study, the effects of myocardial reperfusion-induced δPKC translocation on mitochondrial function were assessed. Mitochondria isolated from hearts that had undergone ischemia (30min) followed by reperfusion (15min) exhibited a significant increase in the rate of superoxide anion (O 2 − ) generation. This was associated with the translocation of δPKC to the mitochondria within the first 5min of reperfusion. δPKC translocation occurred exclusively during reperfusion and could be mimicked by infusion of intact hearts with H 2 O 2 suggesting redox-dependent activation during reperfusion. Infusion of a peptide inhibitor (δV 1-1 ) specific to the δ-isoform of PKC significantly reduced reperfusion-induced increases in mitochondrial O 2 − generation. Finally, the decline in mitochondrial respiratory activity evident upon prolonged reperfusion (120min) was completely prevented by inhibition of δPKC translocation. Thus, δPKC represents a cytosolic redox-sensitive molecule that plays an important role in amplification of O 2 − production and subsequent declines in mitochondrial function during reperfusion.