Mitochondria maintain tight regulation of inner mitochondrial membrane (IMM) permeability to sustain ATP production. Stressful events cause cellular calcium (Ca²⁺) dysregulation followed by rapid loss of IMM potential known as permeability transition (PT), which produces osmotic shifts, metabolic dysfunction, and cell death. The molecular identity of the mitochondrial PT pore (mPTP) was previously unknown. We show that the purified reconstituted c-subunit ring of the F_O of the F₁F_O ATP synthase forms a voltage-sensitive channel, the persistent opening of which leads to rapid and uncontrolled depolarization of the IMM in cells. Prolonged high matrix Ca²⁺ enlarges the c-subunit ring and unhooks it from cyclophilin D/cyclosporine A binding sites in the ATP synthase F₁, providing a mechanism for mPTP opening. In contrast, recombinant F₁ beta-subunit applied exogenously to the purified c-subunit enhances the probability of pore closure. Depletion of the c-subunit attenuates Ca²⁺-induced IMM depolarization and inhibits Ca²⁺ and reactive oxygen species-induced cell death whereas increasing the expression or single-channel conductance of the c-subunit sensitizes to death. We conclude that a highly regulated c-subunit leak channel is a candidate for the mPTP. Beyond cell death, these findings also imply that increasing the probability of c-subunit channel closure in a healthy cell will enhance IMM coupling and increase cellular metabolic efficiency.