c-Ski, originally identified as an oncogene product, induces myogenic differentiation in nonmyogenic fibroblasts through transcriptional activation of muscle regulatory factors. Although c-Ski does not bind to DNA directly, it binds to DNA through interaction with Smad proteins and regulates signaling activities of transforming growth factor-beta (TGF-beta). In the present study, we show that c-Ski activates the myogenin promoter independently of regulation of endogenous TGF-beta signaling. Expression of myogenin is regulated by a transcription factor complex containing proteins of the MyoD family and the myocyte enhancer factor 2 (MEF2) family. c-Ski acts on the MyoD-MEF2 complex and modulates the activity of MyoD in myogenin promoter regulation. Interestingly, histone deacetylase (HDAC) inhibitors up-regulated basal activity of transcription from a MyoD-responsive reporter, although c-Ski failed to further augment this transcription in the presence of HDAC inhibitors. c-Ski is observed both in the cytoplasm and in the nucleus, but its nuclear localization is required for myogenic differentiation. We conclude that c-Ski induces myogenic differentiation through acting on MyoD and inhibiting HDAC activity in the nucleus of myogenic cells.