In response to sustained increase in contractile activity, mammalian skeletal muscle undergoes adaptation with enhanced mitochondrial biogenesis and fiber type switching. The peroxisome proliferator activated receptor co-activator-1 (PGC-1) has recently been identified as a key regulator for these adaptive processes. To investigate the sequence elements in the PGC-1 gene that are responsible for activity-dependent transcriptional activation, we have established a unique system to analyze promoter activity in skeletal muscle of living mice. Expression of PGC-1-firefly luciferase reporter gene in mouse tibialis anterior (TA) muscle transfected by electric pulse-mediated gene transfer was assessed repeatedly in the same muscle by optical bioluminescence imaging analysis before and after low-frequency (10 Hz) motor nerve stimulation. Nerve stimulation (2 h) resulted in a transient 3-fold increase (P<0.05) in PGC-1 promoter activity along with a 1.6-fold increase (P<0.05) in endogenous PGC-1 mRNA. Mutation of two consensus myocyte enhancer factor 2 (MEF2) binding sites (-2901 and -1539) or a cAMP response element (CRE) (-222) completely abolished nerve stimulation-induced increase in PGC-1 promoter activity. These findings provide direct evidence that contractile activity-induced PGC-1 promoter activity in skeletal muscle is dependent on the MEF2 and the CRE sequence elements. The experimental methods used here have general applicability to studies of gene regulation in muscle.