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 (), 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 , signaling the completion of ADP phosphorylation, was used to evaluate the rate of ATP synthesis during repeated ADP pulses. In mitochondria with decreased ability to support , 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 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 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.