A cell line derived from the murine rhabdomyosarcoma BW10139 (Dexter, Cancer Res. 37: 3136, 1977) was subcloned and examined with respect to growth and myogenic characteristics in the presence and absence of 1 mM butyrate. Without butyrate, these cells behave as typical transformed cells: they grow rapidly and chaotically, do not form multinucleated muscle fibers and have little or no creatine kinase activity. In the presence of 1 mM sodium butyrate or butyric acid, growth slows, cells become arranged in whorl patterns, and creatine kinase activities increase to levels comparable to those found in normal chick myoblasts immediately prior to cell fusion. The increase in creatine kinase activity is detectable within 2 h exposure to butyrate, reaches a maximum by 24 h, and the elevated level can be maintained for at least six weeks. The induction is reversible upon sequential addition, deletion, and readdition of butyrate to the culture medium. Isoenzyme analyses demonstrated that only the BB form of creatine kinase is induced; MM creatine kinase was not detected. Although formation of multinucleated cells increases after exposure to butyrate, no typical myotubes form. The results suggest that this rhabdomyosarcoma cell line can, under appropriate conditions, re-express some properties characteristic of skeletal muscle, but not the complete muscle phenotype.