Intracellular Ca(2+) overload has been considered a common pathological precursor of pancreatic injury. In this study, the effects of melatonin on Ca(2+) mobilization induced by cholecystokinin octapeptide (CCK-8) in freshly isolated mouse pancreatic acinar cells have been examined. Changes in intracellular free Ca(2+) concentration were followed by single cell fluorimetry. For this purpose, cells were loaded with the Ca(2+)-sensitive fluorescent dye fura-2-acetoxymethyl ester. In order to evaluate the contribution of Ca(2+) transport at the plasma membrane, at the endoplasmic reticulum (ER) or at the mitochondria, cells were incubated with CCK-8 alone or in combination with LaCl3, thapsigargin (Tps), or FCCP to, respectively, uncouple Ca(2+) transport at these localizations. The experiments were performed in the absence or in the presence of melatonin in combination with the stimuli mentioned. Our results show that the total Ca(2+) mobilization evoked by CCK-8 was attenuated by a 30% in the presence of 100 µM melatonin compared with the responses induced by CCK-8 alone. Upon inhibition of Ca(2+) transport into the ER by Tps, Ca(2+) mobilization was also reduced in the presence of melatonin. In the presence of LaCl3 plus melatonin, the total Ca(2+) mobilization induced by CCK-8 was significantly decreased, compared with the response obtained without melatonin but in the presence of LaCl3. No major differences were found when the cells were incubated with CCK-8 or Tps alone or in combination with LaCl3 plus melatonin and FCCP, compared with the responses obtained in the absence of FCCP. The initial Ca(2+) release from intracellular stores evoked by CCK-8 or Tps was not significantly reduced in the presence of melatonin. The effect of melatonin could be explained on the basis of a stimulated Ca(2+) transport out of the cell through the plasma membrane and by a stimulation of Ca(2+) reuptake into the ER. Accumulation of Ca(2+) into mitochondria might not be a major mechanism stimulated by melatonin. We conclude that melatonin alleviates intracellular Ca(2+) accumulation, a situation potentially leading to cell damage in the exocrine pancreas.