Phorbol-12-myristate-13-acetate (PMA) in some systems causes inhibition of growth; in others, PMA exerts a mitogenic effect. We have previously reported that the PMA-induced growth arrest and differentiation in leukemic cells are associated with a rapid increase in phosphorylation-dephosphorylation of two cytosolic proteins, phosphoprotein with molecular weights of 17,000 to 20,000 (pp17–20) and phosphoprotein with a molecular weight of 27,000 (pl ∼ 5.5). The phosphorylation of these proteins was found to be catalyzed directly by cyclic adenosine 3′:5′-monophosphate-dependent protein kinase. To elucidate whether these phosphorylation events are specific to certain cellular functions induced by PMA, we compared the effect of PMA on phosphorylation of proteins in several cell systems. We found that, 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 Mr ∼80,000 (pl ∼ 5) and Mr 20,000 (pl ∼ 5.2) was markedly increased. We provide evidence to suggest that the Mr 20,000/pl 5.2 protein might be myosin light chain (Mr 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 chain (Mr 20.000/pl 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. These results suggest that phosphorylation of different proteins and possibly activation of different protein kinases mediate the effects of PMA on inhibition versus 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.