The possible role of protein kinase C activation in the inhibitory action of cholinergic transmitters on the slow Ca-dependent afterhyperpolarizing current (IAHP) in hippocampal CA3 pyramidal neurons was investigated using hippocampal slice cultures. IAHP was inhibited reversibly by methacholine (100–600 nM) and irreversibly by the protein kinase C activator, phorbol-12,13-dibutyrate (PDBu, 10 nM to 1 μM). The inhibitory action of PDBu was antagonized by prior (15–60 min) exposure to staurosporin (1 μM). In contrast, the inhibitory effect of methacholine on IAHP was not reduced after up to 3 h of exposure to this compound. In addition, methacholine produced a reversible inward current at the holding potential, which was augmented by staurosporin. However, prior exposure to PDBu reduced the effect of methacholine on IAHP and occluded the methacholine-induced inward current. This effect of PDBu was also observed in the presence of staurosporin, suggesting that it might be exerted through a protein kinase C-independent pathway. Noradrenalin (2–5 μM) and 8-bromo cyclic adenosine 3′,5′monophosphate (8-Br-cAMP, 1 mM) also produced a reversible block of IAHP. Their action was antagonized by staurosporin, probably via its effect on protein kinase A. Thus the present experiments suggest that the action of muscarinic agonists on IAHP cannot be explained by an effect on protein kinase C, but support a role for protein kinase A in mediating the action of noradrenalin.