The mitochondrial permeability transition pore (MPTP) plays a key role in cell death, yet its molecular identity remains uncertain. Although knock-out studies have confirmed critical roles for both cyclophilin-D (CyP-D) and the adenine nucleotide translocase (ANT), given a strong enough stimulus MPTP opening can occur in the absence of either. Here we provide evidence that the mitochondrial phosphate carrier (PiC) may also be a critical component of the MPTP. Phenylarsine oxide (PAO) was found to activate MPTP opening in the presence of carboxyatractyloside (CAT) that prevents ANT binding to immobilized PAO. Only four proteins from solubilized CAT-treated beef heart inner mitochondrial membranes bound to immobilized PAO, one of which was the PiC. GST-CyP-D pull-down and co-immunoprecipitation studies revealed CsA-sensitive binding of PiC to CyP-D; this increased following diamide treatment. Co-immunoprecipitation of the ANT with the PiC was also observed but was insensitive to CsA treatment. N-ethylmaleimide and ubiquinone analogues (UQ(0) and Ro 68-3400) inhibited phosphate transport into rat liver mitochondria with the same concentration dependence as their inhibition of MPTP opening. UQ(0) and Ro 68-3400 also induced the "m" conformation of the ANT, as does NEM, and reduced the binding of both the PiC and ANT to the PAO column. We propose a model for the MPTP in which a calcium-triggered conformational change of the PiC, facilitated by CyP-D, induces pore opening. An interaction of the PiC with the ANT may enable agents that bind to either transporter to modulate pore opening.