The transcriptional co-activator PGC-1α is thought to play an important role in exercise-induced skeletal muscle mitochondrial biogenesis. Classically, PGC-1α is known to regulate and coordinate mitochondrial biogenesis by activating transcription of nuclear-encoded mitochondrial genes. Recently, PGC-1α has been shown to reside in mitochondria; however the physiological consequence of mitochondrial PGC-1α remains unknown. PURPOSE: We aimed to determine the effect of an acute bout of endurance exercise on mitochondrial PGC-1α content, mitochondrial DNA (mtDNA) transcription, and mtDNA copy number. METHODS: C57Bl/6J wild-type mice were randomly assigned to either sedentary (SED) or forced-endurance exercise (END; treadmill run @ 15 m/min for 90 min) group (N = 7/group). The endurance exercise group was sacrificed immediately following a single bout of exercise. We analyzed skeletal muscle (quadriceps femoris) for mRNA expression of nuclear DNA-encoded (PGC-1α, Tfam, cytochrome c, ALAS) and mtDNA-encoded (ND1, ND4, and COX subunit -I and -II) transcripts. Mitochondrial fractions prepared from quadriceps femoris were analyzed for PGC-1α protein content and mtDNA copy number. RESULTS: An acute bout of endurance exercise increased mRNA expression of PGC-1α, Tfam, cytochorome c, ALAS, ND1, ND4, COX-I and II by ∼2-4 fold in skeletal muscle of END mice (P < 0.05 for all transcripts). Endurance exercise increased mitochondrial PGC-1· content by ∼4 fold (P < 0.01). The mtDNA-co-immunoprecipitation assays indicated increased binding of PGC-1α to Tfam in mitochondrial nucleiods in response to an acute bout of endurance exercise (P < 0.05 vs. SED). Acutely, endurance exercise did not alter mtDNA copy number. CONCLUSION: We conclude that an acute bout of endurance exercise increases mitochondrial PGC-1αcontent which may function as a transcriptional co-activator for mtDNA transcription, without acutely altering mtDNA replication. These findings indicate that PGC-1α may function to co-ordinate both nuclear- and mtDNA-encoded gene expression to promote net mitochondrial biogenesis in response to exercise. Supported by Canadian Institutes of Health Research - Institute of Aging.