The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated the synthesis of sphingomyelin (CerPCho) from a [14C]choline-labelled phosphatidylcholine (PtdCho) pool in NIH 3T3 cells. Maximal stimulation (68%) of CerPCho synthesis, accompanied by an increase (38%) in its cellular content, required only 2 nM TPA. Higher concentrations of TPA (2-100 nM) had progressively less effect on CerPCho synthesis which correlated with increased hydrolysis of precursor PtdCho. In cells transformed with human or mouse A-raf carrying retroviruses TPA-stimulated PtdCho hydrolysis, but not CerPCho synthesis, suggesting independent regulation of these processes by the TPA-stimulated signal transduction system.