Type beta transforming growth factor (TGF beta) has been shown to be both a positive and negative regulator of cellular proliferation and differentiation. The effects of TGF beta also are cell-type specific and appear to be modulated by other growth factors. In the present study, we examined the potential of TGF beta for control of myogenic differentiation. In mouse C-2 myoblasts, TGF beta inhibited fusion and prevented expression of the muscle-specific gene products, creatine kinase and acetylcholine receptor. Differentiation of the nonfusing muscle cell line, BC2Hl, was also inhibited by TGF beta in a dose-dependent manner (ID50 approximately 0.5 ng/ml). TGF beta was not mitogenic for either muscle cell line, indicating that its inhibitory effects do not require cell proliferation. Inhibition of differentiation required the continual presence of TGF beta in the culture media. Removal of TGF beta led to rapid appearance of muscle proteins, which indicates that intracellular signals generated by TGF beta are highly transient and require continuous occupancy of the TGF beta receptor. Northern blot hybridization analysis using a muscle creatine kinase cDNA probe indicated that TGF beta inhibited differentiation at the level of muscle-specific mRNA accumulation. These results provide the first demonstration that TGF beta is a potent regulator of myogenic differentiation and suggest that TGF beta may play an important role in the control of tissue-specific gene expression during development.