Significance of cyclophiline D for functioning of the mitochondrial permeability transition pore (mPTP) has been debated (Basso et al., 2005, JBC 280:18558). Therefore, different methods for demonstrating the action of the mPTP were employed comparing mitochondrial activity of wildtype (WT) and of Ppif-/- mice (ko-mice). We measured oxygen consumption of brain mitochondria, changes of their mitochondrial membrane potential (DY) with Safranin O, their Ca-retention capacity with Ca-green, and single-channel activity of the mPTP of liver mitochondria. A sucrose medium (200 mM sucrose, 10 mM Tris, 1 mM KH₂PO₄, 10 mM EGTA, 10 mM glutamate and 2 mM malate; pH 7.2) was used except for patch-clamp measurements which were done in a HEPES-buffered isotonic KCl solution. Respirometry demonstrates that in ko-mice, similar to WT-mitochondria, the complex I-related flux is about 1.4 times that related to complex II. The complex I-related flux was more pronouncedly reduced upon application of 0.33 Ca²⁺ ([Ca²⁺] and [CsA] in mmol/mg) than the complex II-related flux. 0.03 Cyclosporin A (CsA) did not normalize the Ca²⁺-reduced fluxes. In WT-mice, however, the Ca²⁺-reduced fluxes were normalized when Ca²⁺ and CsA were given together. DY collapsed after application of 0.2 Ca²⁺ in liver mitochondria of WT and after 0.4 Ca²⁺ in ko-mice. Similarly, the Ca²⁺ concentration necessary for a collapse of DY in brain mitochondria was lower in WT than in ko-mice. Almost no difference in the required Ca²⁺-concentrations was detected after application of 0.2 CsA in both types of mice. The Ca²⁺-retention capacity was 0.3 in WT brain mitochondria and 0.5 in ko-mice. In liver these values were 0.2 and 0.8. Ca²⁺-retention capacity was increased in WT by application of 0.2 CsA while it was rather reduced by CsA in the ko-mice. The open probability (Po) of the mPTP as measured in single-channel experiments in a high Ca²⁺-concentration (200 mM) vs the CsA-concentration was shifted to the right in ko-mice mitochondria as compared with WT. These results are in agreement with the presence of a mPTP in Ppif-/- mice that is still functioning though with lower sensitivity for CsA and Ca²⁺.