We have studied the effects of human leukocyte interferon produced in bacteria and diterpene phorbol ester tumor promoters on differentiation of normal human myoblast cultures derived from mature skeletal muscle. Interferon (100-5,000 units/ml) induced an acceleration of myotube formation and creatine kinase (CK; EC 22.214.171.124) isoenzyme transition from CK-BB to CK-MM. Heat-inactivated or trypsin-treated interferon did not affect the differentiation process. In contrast, the potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA), but not its inactive structural analogues phorbol and 4 alpha-phorbol 12,13-didecanoate, caused a dose-dependent (0.01-100 ng/ml) inhibition of myotube formation and CK isoenzyme transition. Neither interferon nor TPA had a significant effect on myoblast proliferation prior to fusion, and the cloning efficiencies were similar as well. Opposing effects of interferon and TPA were also demonstrated by simultaneous application of these agents to the cultures. These studies suggest that some of the antitumor effects of interferon may relate to its capacity to modulate cellular differentiation.