The impact of the mitochondrial permeability transition (MPT) on cellular physiology is well characterized. In contrast, the composition and mode of action of the permeability transition pore complex (PTPC), the supramolecular entity that initiates MPT, remain to be elucidated. Specifically, the precise contribution of the mitochondrial F1FO ATP synthase (or subunits thereof) to MPT is a matter of debate. We demonstrate that F1FO ATP synthase dimers dissociate as the PTPC opens upon MPT induction. Stabilizing F1FO ATP synthase dimers by genetic approaches inhibits PTPC opening and MPT Specific mutations in the F1FO ATP synthase c subunit that alter C-ring conformation sensitize cells to MPT induction, which can be reverted by stabilizing F1FO ATP synthase dimers. Destabilizing F1FO ATP synthase dimers fails to trigger PTPC opening in the presence of mutants of the c subunit that inhibit MPT The current study does not provide direct evidence that the C-ring is the long-sought pore-forming subunit of the PTPC, but reveals that PTPC opening requires the dissociation of F1FO ATP synthase dimers and involves the C-ring.