[H₂O₂], a member of reactive oxygen species (ROS), is known to be involved in the mediation of physiological functions in a variety of cell types. However, little has been known about the physiological role of H₂O₂in exocrine cells. Therefore, in the present study, the effect of H₂O₂on cholecystokinin (CCK)-evoked Cα²+ mobilization and amylase release was investigated in rat pancreatic acinar cells. Stimulation of the acinar cells with sulfated octapeptide form of CCK (CCK-8S) induced biphasic increase in amylase release. Addition of 30μM H₂O₂ enhanced amylase release caused by 10 pM CCK-8S, but inhibited the amylase release induced by CCK-8S at concentrations higher than 100 pM. An ROS scavenger, 10 μM Mn(III)tetrakis(4-benzoic acid)porphyrin chloride, increased amylase release caused by CCK-8S at concentrations higher than 100 pM, although lower concentrations of CCK-8S-induced amylase release was not affected. To examine whether the effect of H₂O₂on CCK-8S-induced amylase release was exerted via modulation of intracellular Cα²+ signaling, we measured the changes in intracellular Cα²+ concentration ([Cα²+]_i) in fura-2 loaded acinar cells. Although 30 μM H₂O₂did not induce any increase in([Cα²+]_i by itself, it increased the frequency and amplitude of([Cα²+]_i oscillations caused by 10 pM CCK-8S. However, 30μM H₂O₂had little effect on 1 nM CCK-8S-induced increase in [Cα²+]_i. ROS scavenger, 1 mM N-acetylcysteine, did not affect [Cα²+]_i changes induced by 10 pM or 1 nM CCK-8S. Therefore, it was concluded that 30 μM H₂O₂ enhanced low concentration of CCK-8S-induced amylase release probably by increasing Cα²+]_i oscillations while it inhibited high concentration of CCK-8S-induced amylase release.