The myogenic basic helix-loop-helix (myo-bHLH) proteins are a family of transcriptional regulators expressed in myoblasts and differentiated skeletal muscle. Ectopic expression of myo-bHLH regulators transdetermines some fibroblast cell lines into myoblasts, which exit the cell cycle and differentiate into skeletal muscle when cultured in low mitogen medium. While members of the myo-bHLH family have been shown to function as transcriptional activators in differentiating muscle, the molecular basis of their function in proliferating myoblasts has not been elucidated. In this report, we present evidence that MyoD functions as a transcriptional repressor in myoblasts. We show that transcription from a cyclin B1 promoter construct is repressed in proliferating myoblasts and that repression is mediated by a pair of MyoD binding sites. We also show that transcription from the cyclin B1 promoter is repressed in proliferating C3H10T1/2 cells by ectopic expression of MyoD. These results demonstrate that MyoD can repress transcription of specific genes in proliferating cells, a novel function that may be important to maintenance of the myogenic phenotype and to cell cycle regulation in myoblasts.