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