Within 10 min of addition of the tumor promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA) to C3H/10T1/2 mouse embryo fibroblasts, there is a two-fold increase in the level of cellular 1,2-diacylglycerol levels compared to controls. This increase in 1,2-diacylglycerol is dependent on the concentration of TPA added to the cell culture medium. The ability of macrocyclic diterpenes to induce 1,2-diacylglycerol accumulation correlated with their tumor promoting activity except for mezerein. The accumulation of 1,2-diacylglycerol in response to TPA was not blocked by a concentration of cycloheximide sufficient to inhibit protein synthesis by 95%. These data support our previous suggestion that TPA activates a phospholipase C. During the same time period, TPA increased protein phosphorylation in both quiescent and growing cells. Proteins of mol. wt. approximately 50 000, 45 000, 35 000 and 27 000 are markedly phosphorylated in response to TPA in both growing and quiescent cultures. The relationship of these phosphorylated proteins to a Ca2+ phospholipid activated protein kinase remains to be determined.