C3H/10T 1/2 cells were induced to differentiate into muscle cells by treatment with 5-azacytidine, and the effects of tumor promoters, nonpromoters, and inhibitors of tumor promotion on this induced differentiation were investigated. Cell morphology was dramatically changed within 30 min after treatment with phorbol ester-related tumor promoters and mezereine but not with other tumor promoters. There was a good correlation between the tumor-promoting activity of the compounds and their inhibitory action on differentiation except in case of phenobarbital; this promoter of liver carcinogenesis did not inhibit differentiation. Inhibitors of tumor promotion, dexamethasone, fluocinolone acetonide, retinoic acid, and antipain, also inhibited the 5-azacytidine-induced differentiation with 12-0-tetradecanoylphorbol-13-acetate, a potent tumor promoter, and simultaneously with one of those inhibitors of tumor promotion, the inhibitory action of 12-0-tetradecanoylphorbol-13-acetate was not affected. From these facts, it is evident that using a single phenomenon produced by one class of tumor promoter as a criterion for screening environmental tumor promoters in not justifiable.