DD3 cells are a myoblast line generated by stable transfection of C3H10T1/2 fibroblasts with the bovine myf5 cDNA (bmyf) fused to the dexamethasone-inducible MMTV promoter. After treating proliferating cells with dexamethasone, bmyf transcripts were induced approximately fivefold within 1.5-2.5 h. Induction of bmyf was followed 4 h later by a similar increase in myogenin transcripts which was dependent on protein synthesis. The elevated level of myogenin transcripts in dividing cells was comparable to that observed in DD3 myoblasts after differentiation. The 4-h lag before the myogenin response suggested involvement of an intermediate factor. However, one possible factor, myocyte-specific enhancer binding factor (MEF-2) (a known enhancer of myogenin promoter activity) was neither detectable nor inducible by dexamethasone in proliferating cells. myoD transcripts were barely detectable and uninducible in proliferating cells but strongly upregulated during differentiation. There was also a transient twofold increase in mrf4 transcripts by dexamethasone treatment in dividing cells, while no changes were detected in the levels of Id, E12, or TnC messages. The mouse myf5 gene was silent and uninducible in DD3 cells under proliferating and differentiating conditions. We conclude that ectopic expression of MMTV-bmyf led to activation of three of the endogenous myogenic factor genes of which only myogenin showed a rapid and sustained response to dexamethasone-mediated induction of bmyf in dividing cells.