Skeletal muscle gene expression is dependent on combinatorial associations between members of the MyoD family of basic helix-loop-helix (bHLH) transcription factors and the myocyte enhancer factor 2 (MEF2) family of MADS-box transcription factors. The transmembrane receptor Notch interferes with the muscle-inducing activity of myogenic bHLH proteins, and it has been suggested that this inhibitory activity of Notch is directed at an essential cofactor that recognizes the DNA binding domains of the myogenic bHLH proteins. Given that MEF2 proteins interact with the DNA binding domains of myogenic bHLH factors to cooperatively regulate myogenesis, we investigated whether members of the MEF2 family might serve as targets for the inhibitory effects of Notch on myogenesis. We show that a constitutively activated form of Notch specifically blocks DNA binding by MEF2C, as well as its ability to cooperate with MyoD and myogenin to activate myogenesis. Responsiveness to Notch requires a 12-amino-acid region of MEF2C immediately adjacent to the DNA binding domain that is unique to this MEF2 isoform. Two-hybrid assays and coimmunoprecipitations show that this region of MEF2C interacts directly with the ankyrin repeat region of Notch. These findings reveal a novel mechanism for Notch-mediated inhibition of myogenesis and demonstrate that the Notch signaling pathway can discriminate between different members of the MEF2 family.