Staurosporine was previously shown to mobilize Ca2+ from the thapsigargin-insensitive Ca2+ store in rat submandibular acinar cells. However, the nature of the store is not yet known. Therefore, in the present study, the staurosporine-releasable intracellular Ca2+ store was characterized. Staurosporine increased the cytosolic Ca2+ concentration ([Ca2+]c) after the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store was depleted. Ionomycin caused only small increases in [Ca2+]c after the depletion of the IP3-sensitive Ca2+ store, whereas ionomycin+monensin caused large increases. However, ionomycin+monensin did not increase [Ca2+]c when added after [Ca2+]c was increased by staurosporine, indicating that the acidic Ca2+ store was the main source of Ca2+. The acidic Ca2+ store appeared to be associated with secretory granules, since ionomycin+monensin- and staurosporine-induced [Ca2+]c increases were significantly reduced when the acinar cells were degranulated. The effect of staurosporine on [Ca2+]c was mimicked by other protein kinase C inhibitors. Therefore, we conclude that staurosporine mobilizes Ca2+ from secretory granules, probably through the inhibition of protein kinase C in rat submandibular acinar cells.