We have previously described an PKC interaction with cytochrome oxidase (CO) subunit IV which correlates with enhanced CO activity and cardiac ischemic preconditioning (PC). We therefore, investigated the effects of PC and ischemia/reperfusion (IR) injury on CO subunit levels in an anesthetized rat coronary ligation model. Homogenates prepared from the left ventricular regions at risk (RAR) and not at risk (RNAR) for IR injury were fractionated into cell soluble (S), 600 x g low speed centrifugation (L), gradient-purified mitochondrial (M), and 100,000 x g particulate (P) fractions. In RAR tissue, PC (2 cycles of 5 min ischemia / 5 min reperfusion) decreased the COI subunit in the P fraction (~29 % of total cellular COI) suggesting changes in interfibrillar (IFM) mitochondria. Following 30 minutes of ischemia and 120 minutes of reperfusion, total COI levels decreased in the RAR by 72 %. Subunit Va, was also down-regulated following prolonged IR in the RAR by 42%. PC administered prior to IR reduced the loss of COI in the M and P fractions ~30 % and prevented COVa losses completely. We observed no losses in subunits Vb and VIIa following IR alone, however, significant losses occurred when PC was administered prior to prolonged IR. Delivery of a cell-permeable PKC translocation inhibitor (V1-2) to isolated rat hearts prior to prolonged IR dramatically increased COI loss suggesting that PKC protects COI levels. We propose that additional measures to protect CO subunits when co-administered with PC may improve its cardio-protection following IR injury.