The formation of inositol phosphates and the changes in free intracellular Ca2+ ([Ca2+]i) in isolated rabbit gastric mucous cells during cholinergic stimulation were examined and the potential role of inositol phosphate turnover and [Ca2+]i in gastric mucus secretion evaluated. Rabbit chief and parietal cells were studied for comparison. The formation of [3H]inositol phosphates in mucous, chief, and parietal cells was stimulated in a time- and concentration-dependent fashion by acetylcholine (ACh). The ACh-induced initial [Ca2+]i peak was maximally (10(-4) M ACh) 199 +/- 8% of basal in mucous cells, 427 +/- 20% in chief, and 455 +/- 31% in parietal cells and was followed by a lower-level plateau in mucous and parietal cells but by a more rapid decline in chief cells. As in parietal and chief cells, the initial [Ca2+]i peak occurred in mucous cells in the absence of external Ca2+. ACh stimulated a mucous cell membrane Ca2(+)-entry mechanism in addition to release of Ca2+ from intracellular stores. The concentration-response relationships for the production of [3H]-inositol phosphates, the initial rise in [Ca2+]i, and the stimulation of glycoprotein secretion by ACh were virtually identical. Suppression of the [Ca2+]i rise by the intracellular Ca2(+)-chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) abolished the secretory response. As with many other secretory cells, gastric mucous cells possess cholinergic receptors that upon stimulation mediate the hydrolysis of phosphoinositides, a release of Ca2+ from intracellular stores, and a stimulation of Ca2+ influx through the plasma membrane.