The effects of submillimolar concentrations of sodium butyrate on the differentiation of cultured chick myoblasts have been studied. The continuous presence of 0.5 mM butyrate inhibited myoblast fusion, creatine kinase (CK) isozyme transition, and synthesis of total RNA and protein until the 4th day of myogenesis, after which the fusion index reached control values and total CK activity was elevated. The latter continued to exhibit daily increases over control levels, largely reflecting activity of the MM-CK muscle-specific isozyme which increased to twice the control level by the 8th day. Similar but less striking patterns of early inhibition followed by stimulation were observed for total protein content and synthesis of total protein and RNA. On the other hand, DNA content was slightly but significantly depressed in treated cultures at all times. Butyrate treatment did not reverse 5'-bromodeoxyuridine (BUdR) inhibition of MM-CK differentiation. It was also noted that continuous treatment with 0.5 mM butyrate prevented the unexplained sporadic deterioration of myotubes sometimes observed at the 4th day.