Excised rat hearts were perfused isovolumically and then made globally ischemic for times varying from 0 to 70 min followed by 50 min of reperfusion. In situ mitochondrial electrical potential gradients (Deltapsi(m)) were measured during reperfusion using the lipophilic cation, 3H-tetraphenylphosphonium. Therefore, it was possible to measure the relationships between mechanical performance, Deltapsi(m), and high energy phosphates as a function of time of ischemia. The absolute value of Deltapsi(m) remained constant and then dropped sharply in parallel with mechanical performance after 35 min of ischemia. Eliminating Ca2+ from the reperfusate medium did not preserve Deltapsi(m) nor increase high energy phosphates during the recovery period. An inhibitor of the mitochondrial permeability transition, cyclosporin A, delayed the fall in Deltapsi(m) but did not eliminate it. The data suggest that the mitochondrial permeability transition plays a role in ischemic cell death but is not triggered by influx of Ca2+ through the plasma membrane.