Mitochondria-mediated apoptosis represents a central process driving age-related muscle loss. However, the temporal relation between mitochondrial apoptotic signaling and sarcopenia as well as the regulation of release of pro-apoptotic factors from the mitochondria has not been elucidated. In this study, we investigated mitochondrial apoptotic signaling in skeletal muscle of rats across a wide age range. We also investigated whether mitochondrial-driven apoptosis was accompanied by changes in the expression of Bcl-2 proteins and components of the mitochondrial permeability transition pore (mPTP). Analyses were performed on gastrocnemius muscle of 8-, 18-, 29- and 37-month-old male Fischer344 x Brown Norway rats (9 per group). Muscle weight declined progressively with advancing age, concomitant with increased apoptotic DNA fragmentation. Cytosolic and nuclear levels of apoptosis inducing factor (AIF) and endonuclease G (EndoG) increased in old and senescent animals. In contrast, cytosolic levels of cytochrome c were unchanged with age. Mitochondrial Bcl-2, Bax and Bid increased dramatically in 37-month-old rats, with no changes in the Bax/Bcl-2 ratio in any of the age groups. Finally, expression of cyclophilin D (CyPD) was enhanced at very old age. Our findings indicate that the mitochondrial caspase-independent apoptotic pathway may play a more prominent role in skeletal muscle loss than caspase-mediated apoptosis.