Cholinergic and adrenergic receptor-stimulated inositide hydrolysis was studied in neuronal and glial cells cultured from brains of 1-day-old Wistar-Kyoto rats. Incubation of the cells with [3H]inositol led to the incorporation of radioactivity specifically into inositol phospholipids. Labeling of the membrane lipids reached a maximum in 2-3 days. Receptor-stimulated breakdown of inositides was determined by following the accumulation of inositol phosphates after incubation of the labeled cells for 60 min with carbachol or norepinephrine in the presence of 10 mM lithium. Carbachol (1 mM) stimulated inositol phosphate production in neurons 30 times higher than that seen in glia. The response stimulated by norepinephrine (75 microM) was 6 times higher in neurons than glia. The response to carbachol was blocked by atropine, and the norepinephrine-induced response was inhibited by prazosin suggesting that the receptors mediating the responses were muscarinic and alpha 1-adrenergic, respectively. These results suggest that muscarinic cholinergic and alpha 1-adrenergic stimulated inositide hydrolysis is primarily a neuronal response and that this biochemical event may be important for transmembrane signaling which occurs during neurotransmission.