PPAR coactivator -1 (PGC-1), a transcriptional coactivator, is selectively expressed in slow-twitch fibers in skeletal muscle. Ectopic expression of PGC-1 gene, either in a cell or in an animal, has been shown to promote fast-to-slow fiber type switch. The expression of PGC-1 in muscle is regulated by MEF2 and FKHR, two transcription factors implicated in terminal muscle differentiation. Here we found that PGC-1 expression was activated during terminal muscle differentiation in both C2C12 and Sol8 myoblasts. Using retrovirus mediated MyoD over-expression in C³H10T1/2 cells, we also demonstrated that MyoD, the master regulator of terminal differentiation, could activate PGC-1 expression in vivo. Our transient transfection results also show that myogenic bHLH proteins, especially MyoD, can activate PGC-1 expression by targeting its promoter. Myogenic bHLH protein target sites on PGC-1 promoter were localized to a short region (-49+2) adjacent to the transcription start site, and 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 proteins mediated activation. However, only one site, E2-box, was directly bound by GST-MyoD protein in electrophoresis mobility shift assays. Our results indicate that myogenic bHLH proteins are not only involved in the lineage determination and terminal differentiation, but also directly implicated in the activation of the key fiber-type and metabolic switch gene PGC-1.