The conversion of skeletal myoblasts to terminally differentiated myocytes is negatively controlled by several growth factors and oncoproteins. In this study, we have investigated the molecular mechanisms by which v-Src, a prototypic tyrosine kinase, perturbs myogenesis in primary avian myoblasts and in established murine C2C12 satellite cells. We determined the expression levels of the cell cycle regulators pRb, cyclin D1 and D3 and cyclin-dependent kinase inhibitors p21 and p27 in v-Src-transformed myoblasts and found that, in contrast to myogenin, they are normally modulated by differentiative cues, implying that v-Src affects myogenesis independent of cell proliferation. We then examined the levels of expression, DNA-binding ability and transcription-activation potentials of myogenic regulatory factors in transformed myoblasts and in myotubes after reactivation of a temperature-sensitive allele of v-Src. Our results reveal two distinct potential modes of repression targeted to myogenic factors. On the one hand, we show that v-Src reversibly inhibits the expression of MyoD and myogenin in C2C12 cells and of myogenin in quail myoblasts. Remarkably, these loci become resistant to activation of the kinase in the postmitotic compartment. On the other hand, we demonstrate that v-Src efficiently inhibits muscle gene expression by repressing the transcriptional activity of myogenic factors without affecting MyoD DNA-binding activity. Indeed, forced expression of MyoD and myogenin allows terminal differentiation of transformed myoblasts. Finally, we found that ectopic expression of the coactivator p300 restores transcription from extrachromosomal muscle-specific promoters.