Introduction: Cardiac ischemia/reperfusion (I/R) injury is accompanied by intracellular acidification that can lead to cytosolic and mitochondrial calcium overload. However, the effect of cytosolic acidification on mitochondrial pH (pH_m) and mitochondrial Ca²⁺ (Ca²⁺_m) handling is not well understood. In the present study, we tested the hypothesis that changes in pH_m during cytosolic acidification can modulate Ca²⁺_m handling in cardiac mitochondria. Methods: pH_m was measured in permeabilized rat ventricular myocytes using confocal microscopy and the pH-sensitive fluorescent probe, carboxy-SNARF-1. The contributions of the mitochondrial sodium hydrogen exchanger (NHE_m) and the potassium hydrogen exchanger (KHE_m) to pH_m regulation were evaluated using acidification and recovery protocols to mimic the changes in pH observed during I/R. Ca²⁺_m transport in isolated mitochondria was measured using spectrophotometry and fluorimetry and _m was measured using a TPP⁺ electrode. Results: Cytosolic acidification (pH 6.8) resulted in acidification of mitochondria. The degree of mitochondrial acidification and recovery was found to be largely dependent on activity of the KHE_m. However, the NHE_m was observed to contribute to the recovery of pH_m following acidification in K⁺-free solutions as well as the maintenance of pH_m during respiratory inhibition. Acidification resulted in mitochondrial depolarization and a decrease in the rate of net Ca²⁺_m uptake, while restoration of pH following acidification increased Ca²⁺_m uptake. Conclusions: These findings are consistent with an important role for cytosolic acidification in determining pH_m and Ca²⁺_m handling in cardiac mitochondria under conditions of Ca²⁺ overload. Consequently, interventions which alter pH_m can limit Ca²⁺_m overload and injury during I/R.