We have studied the properties of the permeability transition pore (PTP) in mitochondria from the liver of mice where the Ppif gene encoding for mitochondrial Cyclophilin D (CyP-D) had been inactivated. Mitochondria from Ppif-/- mice had no CyP-D and displayed a striking desensitization of the PTP to Ca2+, in that pore opening required about twice the Ca2+ load necessary to open the pore in strain-matched, wild-type mitochondria. Mitochondria lacking CyP-D were insensitive to Cyclosporin A (CsA), which increased the Ca2+ retention capacity only in mitochondria from wild-type mice. The PTP response to ubiquinone 0, depolarization, pH, adenine nucleotides, and thiol oxidants was similar in mitochondria from wild-type and Ppif-/- mice. These experiments demonstrate that (i) the PTP can form and open in the absence of CyP-D, (ii) that CyP-D represents the target for PTP inhibition by CsA, and (iii) that CyP-D modulates the sensitivity of the PTP to Ca2+ but not its regulation by the proton electrochemical gradient, adenine nucleotides, and oxidative stress. These results have major implications for our current understanding of the PTP and its modulation in vitro and in vivo.