To investigate the mechanism of phospholipid hydrolysis in pancreatic acinar cells, the effects of Ca2+, guanosine 5′-O-(3-thiotriphosphate) (GTPγS) and cholecystokinin (CCK) on both polyphosphoinositide (PI) and phosphatidylcholine (PC) hydrolysis were studied in rat pancreatic acini per-meabilized with the bacterial toxin, streptolysin-O. When acini were prela-beled with my∼-[3H]inositol, permeabilized, and then incubated with various concentrations of free Ca2+for 15 min, Ca2+stimulated [3H]inositol phosphate release at a concentration of 100 nMand was maximally effective at 100 μM. Both GTPγS and CCK enhanced Ca2+-induced [3H]inositol phosphate release, although these agents had no effect in the absence of Ca2+. At a physiological concentration of Ca2+(100 nM), CCK stimulated [3H]inositol phosphate release which was further enhanced by GTPγS. When acini were similarly prelabeled with [3H]choline before permeabilization, [3H]choline phosphate release was also stimulated by free Ca2+over the concentration range from 100 nMto 10 μM. In contrast to PI hydrolysis, however, neither GTPγS, CCK, or GTPγS plus CCK had an additional effect on [3H]choline phosphate release stimulated by 100 nM-100 μM free Ca2+. Furthermore, Ca2+-induced [3H]choline phosphate release appeared to be due to the redistribution from cell to the medium rather than to an increase in choline phosphate production. Therefore, choline phosphate release following prelabeling with [3H]choline is not useful as an indicator of PC hydrolysis in permeabilized acini.