AbstractThe mitochondrial permeability transition pore (MPTP) has resisted molecular identification for decades. The original model of the MPTP had the adenine nucleotide translocator (ANT) as the inner membrane pore-forming component. Indeed, reconstitution experiments showed that recombinant or purified ANT generates MPTP-like pores in lipid bilayers. This model was challenged when mitochondria from Ant1/2 double null mouse liver still showed MPTP activity. Because mice contain and express 3 Ant genes, here we reinvestigated the genetic basis for the ANTs as comprising the MPTP. Liver mitochondria from Ant1, Ant2, and Ant4 deficient mice were highly refractory to Ca2+-induced MPT, and when also given cyclosporine A, MPT was completely inhibited. Moreover, liver mitochondria from mice with quadruple deletion of Ant1, Ant2, Ant4 and Ppif (cyclophilin D, target of CsA) lacked Ca2+-induced MPT. Finally, inner membrane patch clamping in mitochondria from Ant1, Ant2 and Ant4 triple null mouse embryonic fibroblasts (MEFs) showed a loss of MPT-like pores. Our findings suggest a new model of MPT consisting of two distinct molecular components, one of which is the ANTs and the other of which is unknown but requires CypD.One Sentence SummaryGenetic deletion of Ant1/2/4 and Ppif in mice fully inhibits the mitochondrial permeability transition pore