Myogenesis requires energy production for the execution of a number of regulatory and biosynthesis events. We hypothesized that mitochondrial biogenesis would be stimulated during skeletal muscle regeneration. Tibialis anterior muscles of male Sprague-Dawley rats were injected with 0.75% bupivacaine and removed at 3, 5, 7, 10, 14, 21, or 35 days after injection (n = 5-7/group). Two main periods emerged from the histochemical analyses of muscle sections and the expression of proliferating cell nuclear antigen, desmin, and creatine phosphokinase: 1) activation/proliferation of satellite cells (days 3-14) and 2) differentiation into muscle fibers (days 5-35). The onset of muscle differentiation was accompanied by a marked stimulation of mitochondrial biogenesis, as indicated by a nearly fivefold increase in citrate synthase activity and state 3 rate of respiration between days 5 and 10. Peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1) mRNA level and mitochondrial transcription factor A (mtTFA) protein level peaked on day 10 concurrently with the state 3 rate of respiration. Therefore, transcriptional activation by PGC-1 and mtTFA may be one of the mechanisms regulating mitochondrial biogenesis in regenerating skeletal muscle. Taken together, our results suggest that mitochondrial biogenesis may be an important regulatory event during muscle regeneration.