Peroxisomal proliferator-activated receptor-γ coactivator-1α (PGC-1α), a transcriptional coactivator, is selectively expressed in slow-twitch fibers in skeletal muscle. Ectopic expression of the PGC-1α gene in either a cell or an animal has been shown to promote fast to slow fiber-type switch. The expression of PGC-1α in muscle is regulated by myocyte enhancer factor 2 and Forkhead in rhabdomyosarcoma, two transcription factors implicated in terminal muscle differentiation. In this study we found that PGC-1α expression was activated during terminal muscle differentiation in both C2C12 and Sol8 myoblasts. Using retrovirus-mediated MyoD overexpression in C3H10T1/2 cells, we also demonstrated that MyoD, the master regulator of terminal differentiation, could activate PGC-1α expression in vivo. Our transient transfection results also show that myogenic basic helix-loop-helix (bHLH) proteins, especially MyoD, can activate PGC-1α expression by targeting its promoter. Myogenic bHLH protein target sites on PGC-1α promoter were localized to a short region (-49 to ∼+2) adjacent to the transcription start site, which contains two putative E boxes. Mutation of either site significantly reduced MyoD-mediated transactivation in the cells, suggesting that both sites are required for myogenic bHLH protein-mediated activation. However, only one site, the E2 box, was directly bound by glutathione-S-transferase-MyoD protein in EMSAs. Our results indicate that myogenic bHLH proteins not only are involved in lineage determination and terminal differentiation, but also are directly implicated in activation of the key fiber-type and metabolic switch gene, PGC-1α.