To delineate the role of mitochondrial K+ fluxes in cardioprotection, we investigated the effect of extramitochondrial K+ on the ability of mitochondria to support membrane potential (DeltaPsi), regulate matrix volume, consume oxygen, and phosphorylate ADP under conditions mimicking key elements of ischemia-reperfusion. Isolated energized mitochondria responded to ADP addition with depolarization, increased O2 consumption, and matrix shrinkage. The time required for full recovery of DeltaPsi, signaling the completion of ADP phosphorylation, was used to evaluate the rate of ATP synthesis during repeated ADP pulses. In mitochondria with a decreased ability to support DeltaPsi, the rate of ADP phosphorylation was significantly improved by extramitochondrial K+ > Na+ > Li+, especially at higher buffer osmolarity, which promotes matrix shrinkage. K+-induced improvement in DeltaPsi recovery after ADP pulses was accompanied by more rapid and complete matrix volume recovery and enhanced O2 consumption. Manipulations expected to affect matrix swelling by regulating K+ fluxes or water distribution indicate that matrix volume regulation by external factors becomes increasingly important in mitochondria with decreased ability to support DeltaPsi in the face of a high ADP load. Under these conditions, opening of K+ influx pathways improved mitochondrial function and delayed failure. This may be an important factor in the mechanism of diaxozide-induced cardioprotection.