The effects of compounds affecting gastric acid secretion were studied on the formation of inositol phosphates after prelabelling with [3H]-inositol in enriched gastric parietal cells of the rat, prepared by isopycnic centrifugation with Percoll. In cell preparations with 60 to 70% parietal cells, carbachol (10(-6)-10(-2) M) enhanced the accumulation of [3H]-inositol monophosphate ([3H]-IP1), [3H]-inositol bisphosphate ([3H]-IP2) and [3H]-inositol trisphosphate ([3H]-IP3) in a concentration-dependent manner, an effect which was antagonized by 10(-8) M atropine. Li+ (0.5-30 mM) enhanced the basal and carbachol-induced accumulation of all three [3H]-inositol phosphates, the formation of [3H]-IP1 being more sensitive to Li+ than those of [3H]-IP2 and [3H]-IP3. The concentration of Ca2+ in the incubation medium did not affect the relative stimulation of the accumulation of [3H]-inositol phosphates by carbachol, although the basal formation was higher in the presence of Ca2+ in the medium. In the absence of added Ca2+, the incorporation of [3H]-inositol into phospholipids was increased--an effect which was further enhanced by the addition of EGTA to the medium. Gastrin and pentagastrin (10(-8)-10(-5) M) enhanced the formation of [3H]-inositol phosphates, although they were clearly less effective than carbachol. Histamine (10(-6)-10(-3) M) had no effect of its own, but slightly attenuated the effect of carbachol. Cholecystokinin octapeptide (10(-9)-10(-6) M) slightly increased the formation of [3H]-inositol phosphates. Indomethacin (10(-4) M) had no consistent effect on the basal and carbachol-induced accumulation of [3H]-inositol phosphates, nor did prostaglandin E2 (10(-5) M) modify it. Adrenaline (10(-3) M), 5-hydroxytryptamine (10(-3) M), forskolin (10(-5) M), vasopressin (10(-5) M), angiotensin II (10(-5) M) and bombesin (10(-9)-10(-6) M) were all without effect. We suggest that the hydrolysis of inositol phospholipids may be involved in the signal transduction mechanism by which the activation of the muscarinic and gastrin receptors on the parietal cells leads to Ca2+ mobilization and the stimulation of hydrogen ion secretion.