Real-time optical bioluminescence imaging is a powerful tool for studies of gene regulation in living animals. To elucidate exercise-induced signaling/transcriptional control of the peroxisome proliferator-activated receptor-gamma coactivator-1alpha (Pgc-1alpha) gene in skeletal muscle, we combined this technology with electric pulse-mediated gene transfer to cotransfect the Pgc-1alpha reporter gene with plasmid DNA encoding mutant/deletion forms of putative regulatory factors and, thereby, assess the responsiveness of the promoter to skeletal muscle contraction. We show that each of the myocyte enhancer factor 2 sites on the Pgc-1alpha promoter is required for contractile activity-induced Pgc-1alpha transcription. The responsiveness of the Pgc-1alpha promoter to contractile activity could be completely blocked by overexpression of the dominant-negative form of activating transcription factor 2 (ATF2), the signaling-resistant form of histone deacetylase (HDAC) 5 (HDAC5), or protein kinase D (PKD), but not by HDAC4. These findings provide in vivo evidence for functional interactions between PKD/HDAC5 and ATF2 regulatory factors and the Pgc-1alpha gene in adult skeletal muscle.