In order to evaluate the role of secretagogue-induced changes of cytosolic free calcium concentration ([Ca2+]i) in pepsinogen release, the effects of cholecystokinin-octapeptide (CCK-8) on pepsinogen release and on [Ca2+]i in gastric chief cells isolated from guinea pig stomach were investigated. [Ca2+]i was measured with fura-2, Ca(2+)-sensitive fluorescent probe, using dual-wavelength excitation fluorospectrophotometer. In the presence of extracellular Ca2+, CCK-8 induced a rapid and transient increase in [Ca2+]i followed by a sustained plateau of [Ca2+]i increase which remained 20-50 nM above basal level until antagonist was added. Both in the absence of external Ca2+ and in the presence of inorganic Ca2+ channel blocker Ni2+(2 nM), plateau phase but not initial transient peak of [Ca2+]i was diminished. On the other hand, nicardipine, an organic Ca2+ channel blocker, did not affect the [Ca2+]i transient induced by CCK-8. Time course of CCK-8-stimulated pepsinogen release was biphasic; the initial phase of pepsinogen release was unaffected by Ca2+ removal from the medium but was inhibited by Ca2+ chelation with an intracellular Ca2+ chelator BAPTA/AM, while the late phase of pepsinogen release was dependent on extracellular Ca2+. Artificially introduced Ca2+ into the chief cells with ionomycin elicited the increase in [Ca2+]i and pepsinogen release. However, ionomycin-induced release of pepsinogen was transient and several times smaller than that induced by CCK-8. Ni(2+)-sensitive Ca2+ influx across the plasma membrane was also necessary for reloading of agonist-sensitive Ca2+ pool. These results suggest that the initial, transient increase in [Ca2+]i from the intracellular Ca2+ store mediates the initial pepsinogen release, while the sustained increase in [Ca2+]i which is dependent on Ca2+ influx is responsible for the late phase of pepsinogen release. Factors other than the changes in [Ca2+]i may also be required in producing optimal pepsinogen release.