Phorbol-12-myristate-13-acetate (PMA) in some systems causes inhibition of growth; in others, PMA exerts a mitogenic effect. PMA-induced growth arrest and differentiation in leukemic cells evidently is associated with a rapid increase in phosphorylation-dephosphorylation of 2 cytosolic proteins, phosphoprotein with MW of 17,000-20,000 (pp17-20) and phosphoprotein with a MW of 27,000 (pI .apprx. 5.5). The phosphorylation of these proteins was found to be catalyzed directly by cAMP-dependent protein kinase. To elucidate whether these phosphorylation events are specific to certain cellular functions induced by PMA, the effect of PMA on phosphorylation of proteins was studied in several cell systems. In cells where PMA accelerates growth (NIH/3T3 mouse fibroblasts, JB-6 mouse epidermal cells and human peripheral lymphocytes) as well as in human platelets, the phosphorylation of pp17-20 was only slightly affected by PMA, while phosphorylation of other proteins at MW .apprx. 80,000 (pI .apprx. 5) and MW 20,000 (pI .apprx. 5.2) was markedly increased. The MW 20,000/pI 5.2 protein might be myosin light chain (MW 20,000). In contrast to these cell systems, in the human malignant cell A431 epidermoid carcinoma, the growth of which is inhibited by PMA, the pattern of protein phosphorylation by PMA exhibited striking similarity to that of leukemic cells; phosphorylation of pp17-20 was dramatically increased while the phosphorylation of the putative myosin light (MW 20,000 pI 5.2) was not affected. The identity of pp17-20 in A431 and HL-60 leukemic cells was demonstrated by the unique stability of its phosphoester bond to alkali treatment. Phosphorylation of different proteins and possibly activation of different protein kinases evidently mediate the effects of PMA on inhibition vs. acceleration of growth. Specifically, phosphorylation-dephosphorylation of pp17-20 emerges as a signal which might be preferentially involved in PMA effect in certain neoplastic cells.