We recently reported that treatment of differentiated chick embryo myoblasts in culture with the potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a 2-fold increase in the level of 1,2-diacylglycerol in the plasma membrane fraction within 15-30 min (Grove, R.I. and Schimmel, S.D. (1981) Biochem. Biophys. Res. Commun. 102, 158-164). This system has been characterized further and the metabolic origin and fate of the stimulated diacylglycerol have been investigated. The stimulation of 1,2-diacylglycerol was insensitive to alterations of Ca2+ concentration in the medium and to the presence of inhibitors of Ca2+ flux, protein synthesis and prostaglandin synthesis. The fatty acid composition of the newly formed diacylglycerol was similar to that of phosphatidylcholine. In addition, the glycerol moiety of the diacylglycerol was shown to be derived from a lipid with metabolic turnover similar to that of phosphatidylcholine. The tumor promoter was also found to stimulate rapidly synthesis of phosphatidic acid, phosphatidylinositol and phosphatidylcholine. A possible model is proposed, therefore, in which the tumor promoter stimulates a membrane-associated phospholipase C which generates 1,2-diacylglycerol via the hydrolysis of phosphatidylcholine. The newly formed diacylglycerol is then metabolized back to phosphatidylcholine or to phosphatidic acid and phosphatidylinositol.