The carbachol and adrenaline-mediated accumulation of inositol monophosphate isomers in rat cortical prisms has been studied using a commonly employed experimental protocol involving preincubation with myo-[2- 3H]-inositol and subsequent incubation with agonists in the presence of 10 mM LiCl. Inositol phosphate isomers have been analysed by HPLC and identified by comparison of their elution characteristics with those of commercially available standards and the degradation products of authentic Ins 1,3,4-P 3 and Ins 1,4,5-P 3. Incubation of prelabelled cortical prisms for 1 hr with 10 mM LiCl alone gives rise to accumulation of radioactivity in two inositol monophosphate peaks which co-elute with Ins 1-P and Ins 4-P and one major bisphosphate peak which co-migrates with Ins 1,4-P 2. Most of the monophosphate radioactivity is recovered in the Ins 4-P peak (Ins 1-P/Ins 4-P labelling ratio 0.68). Both carbachol and adrenaline produce dose-dependent increases in the labelling of Ins 1-P and Ins 4-P which are antagonized by atropine and prazosin respectively. However, carbachol produces a larger stimulation of accumulation of both monophosphates and also gives rise to a larger selective increase in the accumulation of Ins 1-P (Ins 1-P/4-P labelling ratio 1.40 in the presence of 1 mM carbachol, 0.98 in the presence of 1 mM adrenaline). Kinetic studies of the carbachol-stimulated increases in inositol mono- and bisphosphate labelling have revealed that, in the early period following carbachol addition (0–5 min), Ins 4-P and Ins 1,4-P 2 are labelled more rapidly than Ins 1-P, whereas the reverse is true at later periods (15–60 min) of the incubations. These observations, coupled with the low levels of labelling of the major Ins 1,3,4-P 3 breakdown products (Ins 1,3-P 2 and Ins 3,4-P 2) compared with that of Ins 1,4-P 2, suggest that large-scale production of Ins 1-P is a comparatively late feature of carbachol-mediated inositol phospholipid metabolism and that, if the Ins 1-P is derived from breakdown of Ins 1,3,4,5-P4 via Ins 1,3,4-P 2, the turnover of Ins 1,3-P 2 + Ins 3,4-P 2 must be approximately one order of magnitude greater than that of the Ins 4-P precursor, Ins 1,4-P 2.