The ability of various receptor agonists and elevated extracellular potassium to initiate inositol phospholipid hydrolysis in various regions of rat brain was examined by using a direct assay which involves prelabeling slices with [3H]inositol and assaying [3H]inositol phosphates ([3H]IPs) in the presence of lithium. Both carbachol and noradrenaline evoked an increase in [3H]IP accumulation in all cerebral regions, although there were marked topographical differences in maximal responsiveness. These marked differences do not seem to be due to regional differences in coupling as similar apparent affinities of full agonists and the relative intrinsic activities of partial agonists were obtained. Both carbachol and noradrenaline responses were antagonized equipotently in all the brain regions tested by the muscarinic and alpha-1 antagonists atropine and prazosin, respectively. However, the putatively selective muscarinic antagonist pirenzepine did show an (approximately 10-fold) apparent selectivity against the carbachol responses elicited in the forebrain regions from those in the pons-medulla. Evaluation of extracellular potassium to 18 mM resulted in an increased production of [3H]IPs in all brain regions except the cerebellum. Incubation of slices with the cholinesterase inhibitor physostigmine (50 microM) and the dihydropyridine Ca++ channel activator BAY-K-8644 (1 microM) greatly enhanced the responses produced by elevated K+ in the forebrain regions but had markedly weaker effects in the hindbrain regions. The elevated K+ response alone and the enhanced response in the presence of BAY-K-8644 were both antagonized significantly by the dihydropyridine antagonist (+)-PN-205-033 in all brain regions, by 70 to 80 and 70 to 95%, respectively.