Loss of neural input to skeletal muscle fibres induces atrophy and degeneration with evidence of mitochondria-mediated cell death. However, the effect of denervation on the permeability transition pore (PTP), a mitochondrial protein complex implicated in cell death, is uncertain. In the present study, the impact of 21 days of denervation on the sensitivity of the PTP to Ca²⁺-induced opening was studied in isolated muscle mitochondria. Muscle denervation increased the sensitivity to Ca²⁺-induced opening of the PTP, as indicated by a significant decrease in calcium retention capacity (CRC: 111 ± 12 vs. 475 ± 33 nmol/mg protein for denervated and sham, respectively). This phenomenon was partly attributable to in vivo mitochondrial and whole muscle Ca²⁺ overload. Cyclosporin A, which inhibits PTP opening by binding to cyclophilin D (CypD), was significantly more potent in mitochondria from denervated muscle and restored CRC to the level observed in mitochondria from sham-operated muscles. In contrast, the Cyp-D independent inhibitor trifluoperazine was equally effective at inhibiting PTP opening in sham and denervated animals and did not correct the difference in CRC between groups. This phenomenon was associated with a significant increase in the content of the PTP regulating protein CypD relative to several mitochondrial marker proteins. Together, these results indicate that Ca²⁺ overload in vivo and an altered expression of CypD could predispose mitochondria to permeability transition in denervated muscles.