In rat brain slices the synthesis of [3H]phosphoinositides and the production of [3H]inositol monophosphate (IP1) induced by norepinephrine (NE) were inhibited by glutamate. Calcium concentrations were varied to test if these inhibitory effects of glutamate were mediated by a calcium-dependent process. Although reducing calcium or addition of the calcium antagonist verpamil reduced the inhibitory effects of glutamate, these results were equivocal because reduced calcium directly decreased agonist-induced [3H]phosphoinositide synthesis. The inhibitory effects of glutamate were mimicked by quisqualate in a dose-dependent manner, but none of a variety of excitatory amino acid receptor antagonists modified the inhibition caused by quisqualate. It is suggested that glutamate activates a quisqualate-sensitive receptor (for which an antagonist is not available) and causes inhibition of phosphoinositide hydrolysis mediated in part by a direct or indirect inhibitory effect of calcium on phosphoinositide synthesis. Modulatory effects of arachidonic acid were examined because glutamate and calcium can activate phospholipase A2. Arachidonic acid caused a rapid and dose-dependent inhibition of [3H]phosphoinositide synthesis and of NE-stimulated [3H]IP1 production. A similar inhibition of the response to carbachol also occurred. The inhibition caused by arachidonic acid was unchanged by addition of inhibitors of cyclooxygenase or lipoxygenase. Activation of phospholipase A2 with melittin caused inhibitory effects similar to those of arachidonic acid. Inhibitors of phospholipase A2 were found to impair phosphoinositide metabolism, likely due to their lack of specificity for phospholipase A2. Further studies were carried out in slices that were prelabelled with [3H]inositol in an attempt to separate modulatory effects on [3H]phosphoinositide synthesis and agonist-stimulated [3H]IP1 production. Several excitatory amino acid agonists inhibited NE-stimulated [3H]IP1 production. This inhibitory interaction could be due to impaired synthesis of [3H]phosphoinositides because, even though the slices were prelabeled, addition of unlabelled inositol reduced NE-stimulated [3H]IP1 production, indicating that continuous regeneration of [3H]phosphoinositides is required. In contrast to the inhibitory effects of the excitatory amino acids, gamma-aminobutyric acid (GABA) enhanced the response to NE in cortical and hippocampal slices. GABA also enhanced the response to carbachol in hippocampal and striatal slices and to ibotenic acid in hippocampal slices. Baclofen potentiated the response to NE similarly to the effect of GABA and baclofen partially blocked the inhibitory effect of arachidonic acid but did not alter that of quisqualate.