By using the polymerase chain reaction to amplify specific regions of the respective cDNAs, we have studied the expression of genes encoding the myogenic regulatory factors Myf5, MyoD, Myogenin, and MRF4 (Myf6, herculin) in cultured mouse muscle cells (inducible and permissive C2 cells and Sol8 cells). These cell lines may represent distinct stages in the progression of determined, or committed, muscle cells toward terminal differentiation. Transcripts for Myf5 were detected at the myoblast stage in all the committed muscle cells tested. Expression of the gene for MyoD appeared to be optional at the myoblast stage (MyoD is present in permissive myoblasts and absent from inducible myoblasts) but, like Myogenin, was found to accompany terminal differentiation. Furthermore, forced expression of MyoD converted inducible C2 cells into permissive cells. Expression of MRF4 was found to follow expression of the three other factors and to occur after the onset of terminal differentiation. Of particular interest was the finding that expression of MRF4 was temporally correlated with expression of the gene for the acetylcholine receptor epsilon-subunit that is characteristic of the adult receptor. In vivo, the only transcripts for myogenic regulatory factors to be detected in 8-day mouse embryos were those for Myf5, while expression of MRF4 followed expression of Myf5, MyoD, and Myogenin in developing limbs. Temporal and phenotypic differences related to the expression of Myf5, MyoD, Myogenin, and MRF4 suggest that these factors fulfil distinct roles in the control of myogenesis.