Myf-6, alternatively called MRF4 or herculin, is a member of a group of muscle-specific transcription factors which also comprises Myf-5, myogenin and MyoD. All family members show distinct expression patterns during skeletal muscle development and can convert a variety of cell lines to myocytes. We disrupted the Myf-6 gene in mice to investigate its functional role in the network of regulatory factors controlling myogenesis. Homozygous mice carrying the disrupted Myf-6 gene show pronounced down-regulation of Myf-5 transcription for reasons presently unknown. Consequently, these mice represent a double knock-out model for Myf-6 and Myf-5. The mutants resemble most of the Myf-5 phenotype with aberrant and delayed early myotome formation and lack of distal rib structures. In addition, we find a reduction in the size of axial muscles in the back. Apart from changes in the pattern of some contractile protein isoforms, the existing myofibers appear fairly normal. This suggests that Myf-6 has no major role in the maturation of myotubes, as previously proposed. Our results provide evidence that skeletal myogenesis can proceed in the absence of two myogenic factors, Myf-5 and Myf-6, therefore they must exert largely non-redundant functions in vivo.