The differential effects of muscarinic agents on inositol phospholipid hydrolysis and the role in this process of putative muscarinic receptor subtypes (M1 and M2) were investigated in three regions of guinea pig brain. Addition of the agonist oxotremorine-M to slices of neostriatum, cerebral cortex, or hippocampus incubated in the presence of myo-[2-3H]inositol and Li+ resulted in a large accumulation of labeled inositol phosphates (733, 376, and 330% of control, respectively). In each tissue, the principal product formed was myo-inositol 1-phosphate (59-86%), with smaller amounts of glycerophosphoinositol and inositol bisphosphate. Only trace amounts of inositol trisphosphate could be detected. Regional differences were observed in the capacity of certain partial agonists to evoke inositol lipid hydrolysis, the most notable being that of bethanechol, which was four times more effective in the neostriatum than in either the cerebral cortex or hippocampus. In addition, the full agonists, oxotremorine-M and carbamoylcholine, were more potent stimulators of inositol phosphate release in the neostriatum than in the cerebral cortex. The putative M1 selective agonist 4-m-chlorophenylcarbamoyloxy-2-butynyl trimethyl ammonium chloride had little stimulatory effect in any brain region, whereas the putative M1 selective antagonist pirenzepine blocked the enhanced release of inositol phosphates with high affinity in the cerebral cortex and hippocampus (Ki = 12.1 and 13.9 nM; "M1") but with a lower affinity in the neostriatum (Ki = 160 nM; "M2"). In contrast to its differential effects on stimulated inositol lipid hydrolysis, no regional differences were observed in the capacity of pirenzepine to displace [3H]quinuclidinyl benzilate, a muscarinic antagonist, bound to membrane fractions. Atropine, an antagonist that does not discriminate between receptor subtypes, inhibited the enhanced release of inositol phosphates with similar affinities in the three regions (Ki = 0.40-0.60 nM). The results indicate that by measurement of inositol lipid hydrolysis, regional differences in muscarinic receptor coupling characteristics become evident. These differences, which are not readily detected by radioligand binding techniques, might be accounted for by either the presence of functionally distinct receptor subtypes, or alternatively, by regional variations in the efficiency of muscarinic receptor coupling to inositol lipid hydrolysis.