Receptor-mediated phosphoinositide hydrolysis was studied in 3H-myo-inositol labelled rat cerebral cortical slices. Several CNS neurotransmitter receptor agonists stimulated the hydrolysis of inositol phospholipid(s). Maximal responses to receptor stimulation showed the order, muscarinic cholinergic > adrenergic > serotonergic > histaminergic. Potassium ion depolarisation and a Ca2+ ionophore also stimulated the accumulation of 3H-inositol phosphate(s). The metabolic sequence of muscarinic receptor-mediated phosphoinositide breakdown was examined in detail. Carbachol stimulated the sustained accumulation (> 45 min.) of 3H-Ins P1, 3H-Ins P1, 3H-Ins P2, 3H-Ins P3 and of a novel 3H-inositol phosphate identified as Ins-1,3,4,5-P4. Kinetic studies showed that muscarinic receptor activation results in the rapid (< 5 sec.) increased accumulation of 3H-Ins P2, 3H-Ins P3 and 3H-Ins P4 while the onset of 3H-Ins P1 accumulation is delayed. Using hplc, the Ins P3 fraction was resolved into two components with the retention times of Ins-1,3,4-P3 and Ins-1,4,5-P3. Stimulated accumulation of Ins-1,3,4-P3 was preceded by that of the other polyphosphates. The probable formation of Ins-1,3,4-P3 via Ins-1,3,4,5-P4 dephosphorylation is discussed. A phospholipid precursor for Ins-1,3,4,5-P4 could not be identified but production of this molecule via an ATP-dependent, Ins-1,4,5-P3 3-kinase was confirmed. Studies of the rates at which the separate 3H-inositol phosphates are hydrolysed in stimulated tissue suggest considerable flux through this kinase reaction and indicate that the majority of the 3H-Ins P1, and 3H-Ins P2 accumulating in response to agonist result from 3H-tris- and 3H-tetrakisphosphate metabolism. Pharmacological data support these conclusions for conditions of both high and low receptor occupancy. Lithium ions markedly affected muscarinic receptor-mediated 3H-inositol phosphate metabolism, dose-dependently potentiating stimulated 3H-Ins P3, and 3H-Ins P2 accumulations while concomitantly attenuating those of 3H-Ins P3 and particularly 3H-Ins P4. The latter effects were half-maximal at 1 mM Li+, exhibited a delayed onset, were not related to receptor desensitization but may be indirect consequences of Ins P1 phosphomonoesterase inhibition. The significance of these actions is discussed in the context of the potential second messenger roles of Ins-1,4,5-P3 and Ins-1,3,4,5-P4.