The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) dramatically modifies the differentiative program of myotubes, developed in culture from chick embryo skeletal myogenic cells. In fact TPA selectively decreases the expression of differentiative parameters with a lag of 8-10 h from its administration to the cultures. We have tested whether the reported effect of TPA depends on the synthesis of specific products during the lag phase of TPA action. The data presented indicate that inhibition of protein synthesis by the use of cycloheximide prevents the appearance of TPA induced inhibition of the expression of differentiative products, such as creatine phosphokinase (CPK) activity and acetylcholine receptors (AChR). Following removal of cycloheximide and reinitiation of normal protein synthesis, the TPA induced inhibitory effect on CPK and AChR appears after a delay of about the same length as the time lag of TPA action. Our results indicate that inhibition of protein synthesis during the lag phase of TPA action prevents the effect of this tumor promoter on myotube differentiative parameters, and suggest that the expression of differentiative traits in cultured myotubes is affected by TPA via a regulatory step implying protein synthesis.