The relative roles of protein kinase C in regulating excitatory amino acid-, cholinoceptor-, and adrenoceptor-stimulated phosphoinositide hydrolysis were studied. Slices of rat hippocampus were prelabeled with [3H]-myo-inositol, and agonist-induced [3H]-phosphoinositide hydrolysis was measured by the formation of [3H]-inositol monophosphate ([3H]-IP) in the presence of lithium ion. Activation of protein kinase C with phorbol 12,13-dibutyrate (PDB) (10(-6) M) completely inhibited ibotenate (IBO) (10(-3) M)-induced [3H]-phosphoinositide hydrolysis. Half-maximal inhibition was observed at about 10(-7) M PDB. Higher concentrations of PDB were required to inhibit stimulation of [3H]-IP by either carbachol (CARB) (10(-3) M) or norepinephrine (NE) (10(-4) M, and only partial inhibition could be attained. Preincubation with staurosporine (STAURO) (10(-5) M) or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) (10(-4) M), inhibitors of protein kinase C, potentiated IBO- but not CARB- or NE-induced stimulation of [3H]-IP. PDB inhibition of IBO- or NE-stimulated [3H]-phosphoinositide hydrolysis was reversed by co-addition of STAURO or H-7. In the case of IBO + STAURO, this reversal was to the potentiated level observed with STAURO alone. Enhanced agonist stimulation and reversal of PDB inhibition were also produced by STAURO when [3H]-phosphoinositide hydrolysis was stimulated by either L-glutamate or quisqualate. These experiments show that direct activation of protein kinase C by PDB leads to inhibition of phosphoinositide hydrolysis mediated by excitatory amino acid receptors, cholinoceptors, or adrenoceptors. However, the enhanced agonist-stimulated phosphoinositide hydrolysis elicited by inhibitors of protein kinase C suggests that, when protein kinase C is indirectly activated, only excitatory amino acids rapidly inhibit further receptor-coupling.