Chronic muscle disuse induced by denervation reduces mitochondrial content and produces muscle atrophy. To investigate the molecular mechanisms responsible for these adaptations, we assessed 1) mitochondrial biogenesis- and apoptosis-related proteins and 2) apoptotic susceptibility and cell death following denervation. Rats were subjected to 5, 7, 14, 21, or 42 days of unilateral denervation of the sciatic or peroneal nerve. Muscle mass and mitochondrial content were reduced by 40-65% after 21 and 42 days of denervation. Denervation-induced decrements in mitochondrial content occurred along with 60% and 70% reductions in transcription factor A (Tfam) and peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha, respectively. After 42 days of denervation, Bax was elevated by 115% and Bcl-2 was decreased by 89%, producing a 16-fold increase in the Bax-to-Bcl-2 ratio. Mitochondrial reactive oxygen species production was markedly elevated by 5- to 7.5-fold in subsarcolemmal mitochondria after 7, 14, and 21 days of denervation, whereas reactive oxygen species production in intermyofibrillar (IMF) mitochondria was reduced by 40-50%. Subsarcolemmal and IMF mitochondrial levels of MnSOD were also reduced by 40-50% after 14-21 days of denervation. The maximal rate of IMF mitochondrial pore opening (V(max)) was elevated by 25-35%, and time to V(max) was reduced by 20-25% after 14 and 21 days, indicating increased apoptotic susceptibility. Myonuclear decay, assessed by DNA fragmentation, was elevated at 7-21 days of denervation. Our data indicate that PGC-1alpha and Tfam are important factors that likely contribute to the reduced mitochondrial content after chronic disuse. In addition, our results illustrate that, despite the reduced mitochondrial content, denervated muscle has greater mitochondrial apoptotic susceptibility, which coincided with elevated apoptosis, and these processes may contribute to denervation-induced muscle atrophy.