The effects of highly purified (greater than 5 X 10(7) IU/mg) murine beta-interferon (IFN) on a mouse myoblast line (MM14DZ) have been investigated to confirm and extend the previous observation that partially purified chicken interferon inhibits differentiation of cultured avian myoblasts (Lough et al., Biochem Biophys Res Commun 109:92, 1982). Cultures treated with 20-2,000 lU IFN/ml medium for 5 days exhibited dose-dependent 1) inhibition of differentiation, as indicated by reduced myotube formation and creatine kinase (CK) activity and 2) increases in DNA content, suggesting that the inhibitory effect was accompanied by continued proliferation of myoblasts. Mock-IFN had no such effects. Based on findings in other systems that IFN inhibits activity of ornithine decarboxylase (ODC), the polyamine products of which are required for myogenesis, the hypothesis that inhibition of differentiation was mediated by an effect of IFN on polyamine metabolism was tested. However, observations that 1) IFN-treated myoblasts retained control levels of ODC activity and 2) exogenous polyamines did not prevent IFN-inhibition did not indicate such a mechanism of action. On the other hand, treatment of control cultures with polyamines alone resulted in potentiation of myogenesis as revealed by precocious myotube formation and a marked increase in CK activity.