The effects of phorbol 12-myristate 13-acetate (PMA) on carbamylcholine (CBC)-induced [3H]cyclic GMP formation in mouse neuroblastoma cells (clone N1E-115) were studied. PMA, but not 4.alpha.-phorbol, suppressed muscarinic receptor-mediated cyclic GMP responses in a time-dependent and a concentration-dependent fashion with an IC50 of 68.8 .+-. 20.2 nM. The inhibitory effects of PMA on CBC-induced cyclic GMP formation were of a mixed competitive and noncompetitive type, being characterized by a depression of maximal cyclic GMP response to CBC and a significant increase in its EC50. PMA also significantly reduced (3H]cyclic GMP formation induced by histamine, without affecting the responses elicited either by sodium azide or the calcium ionophore A23187. Although the inhibitory effects of PMA on CBC-induced cyclic GMP formation were not reversed by washing, these effects were significantly attenuated by H-7 [1-(5-isoquinolinesulfony)-2-methylpiperazine], a protein kinase C inhibitor. PMA had not effect on binding of an antagonist ligand to muscarinic receptors, or on the binding characteristics of CBC to these receptors in intact cells. On the other hand, PMA competed for the specific binding of a labeled phorbol ester in intact cells with a potency similar to that of PMA in inhibiting muscarinic receptor-mediated [3H]cyclic GMP responses.