Previous studies demonstrated that phorbol esters and thyrotropin-releasing hormone (TRH) stimulated phosphatidylcholine synthesis via protein kinase C in GH3 pituitary cells (Kolesnick, R. N. (1987) J. Biol. Chem. 262, 14525-14530). Since phosphatidylcholine may serve as the precursor for sphingomyelin synthesis, studies were performed to assess the effect of protein kinase C on sphingomyelin synthesis. The potent phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), stimulated time- and concentration-dependent incorporation of 32Pi into the head group of sphingomyelin in cells short term labeled with 32Pi and resuspended in medium without radiolabel. TPA (10(-7) M) increased incorporation at a rate 1.4-fold of control after 2 h; EC50 congruent to 2 x 10(-9) M TPA. This correlated closely to TPA-induced phosphatidylcholine synthesis; EC50 congruent to 9 x 10(-10) M TPA. TRH (10(-7) M), which activates protein kinase C via a receptor-mediated mechanism, similarly stimulated 32Pi incorporation into sphingomyelin at a rate 1.5-fold of control; EC50 congruent to 5 x 10(-10) M TRH. This correlated closely with TRH-induced phosphatidylcholine and phosphatidylinositol synthesis; EC50 congruent to 2 x 10(-10) and 1.5 x 10(-10) M TRH, respectively. In cells short term labeled with [3H]palmitate, TRH induced a time- and concentration-dependent reduction in the level of [3H]ceramide and a quantitative increase in the level of [3H]sphingomyelin. Compositional analysis of the incorporated [3H]palmitate revealed that TRH increased radiolabel into both the sphingoid base and the fatty acid moieties of sphingomyelin. Similarly, TRH increased incorporation of [3H] serine into sphingomyelin to 145 +/- 8% of control after 3 h. TPA also stimulated these events. Like the effect of TRH on phosphatidylcholine synthesis, TRH-induced sphingomyelin synthesis was abolished in cells "down-modulated" for protein kinase C. In contrast, TRH-induced phosphatidylinositol synthesis still occurred in these cells. These studies suggest that protein kinase C stimulates coordinate synthesis of phosphatidylcholine and sphingomyelin. This is the first report of stimulation of sphingomyelin synthesis via a cell surface receptor.