Skeletal muscle development is controlled by a family of muscle-specific basic helix-loop-helix (bHLH) transcription factors that activate muscle genes by binding E-boxes (CANNTG) as heterodimers with ubiquitous bHLH proteins, called E proteins. Myogenic bHLH factors are expressed in proliferating undifferentiated myoblasts, but they do not initiate myogenesis until myoblasts exit the cell cycle. We describe a bHLH protein, MyoR (for myogenic repressor), that is expressed in undifferentiated myoblasts in culture and is down-regulated during differentiation. MyoR is also expressed specifically in the skeletal muscle lineage between days 10.5 and 16.5 of mouse embryogenesis and down-regulated thereafter during the period of secondary myogenesis. MyoR forms heterodimers with E proteins that bind the same DNA sequence as myogenic bHLH/E protein heterodimers, but MyoR acts as a potent transcriptional repressor that blocks myogenesis and activation of E-box-dependent muscle genes. These results suggest a role for MyoR as a lineage-restricted transcriptional repressor of the muscle differentiation program.