To discover the relationship between the activation of protein kinase C and the generation of reactive oxygen in the tumor promotion process, we investigated the effects of radical scavengers on diacylglycerol-promoted transformation in BALB/3T3 cells. Superoxide dismutase (SOD) showed inhibitory effects on both 1-oleoyl-2-acetylglycerol (OAG)-promoted and diolein-promoted transformation. Catalase (CT) suppressed the promoting effects of diolein by up to 70%. Mannitol (MT), a hydroxyl radical (.OH) scavenger, inhibited diacylglycerol-promoted transformation dose-dependently. These results suggest that activation of protein kinase C alone is insufficient and that generation of reactive oxygen accompanied by activation of the enzyme is essential to the promotion process in BALB/3T3 cells.