The stimulation of the formation of inositol phosphates by various cholinergic agonists and antagonists was studied in rat brain cortical slices. Incubation of the slices with [3H]inositol led to the incorporation of radioactivity into inositol lipids. The accumulation of inositol phosphates was then followed in the presence of 8 mM lithium which blocks the hydrolysis of inositol phosphate. The release of inositol phosphate was linear up to 15 min when stimulated by 1 mM carbachol. Acetylcholine, muscarine, and methacholine also stimulated the release of inositol phosphates with about the same efficacy as carbachol. Oxotremorine, arecoline, pilocarpine, and bethanechol were not as effective as carbachol at stimulating the accumulation of inositol phosphates. Indicative of partial agonist activity, oxotremorine and pilocarpine inhibited the maximal response induced by carbachol. Muscarinic antagonists atropine, scopolamine, and pirenzepine blocked the stimulation by acetylcholine in contrast to nicotinic antagonists, which had no effect. The brain regional response to carbachol-stimulated inositol phosphate release varied widely with large responses observed in the striatum, cerebral cortex, and hippocampus. Smaller responses were seen in the brainstem, hypothalamus, and cerebellum. Although carbachol stimulated inositol phosphate release in cortical slices in the absence of added calcium, EGTA completely blocked the response. These results suggest that the previously characterized stimulation of the incorporation of 32Pi into phosphatidylinositol by cholinergic agonists in synaptosomes (Fisher, S. K., P. D. Klinger, and B. W. Agranoff (1983) J. Biol. Chem. 258: 7358-7363) is due to the initial hydrolysis of inositol lipids.(ABSTRACT TRUNCATED AT 250 WORDS)