Aberrant cytosolic Ca²⁺ flux in pancreatic acinar cells is critical to pathologic pancreatic zymogen activation observed in acute pancreatitis, but the downstream effectors are not known. In this study, we examined the role of the Ca²⁺ activated phosphatase PP2B, or calcineurin in zymogen activation. Isolated pancreatic acinar cells were stimulated with supraphysiologic caerulein (100 nM) with or without the calcineurin inhibitors FK506 or a cell permeable calcineurin inhibitory peptide (CiP). Chymotrypsin activity was measured as a marker of zymogen activation and percent amylase secretion as a measure of enzyme secretion. Cytosolic Ca²⁺ changes were recorded in acinar cells loaded with the intermediate Ca²⁺-affinity dye fluo-5F using a scanning confocal microscope. A 50% reduction in chymotrypsin activity was observed after pretreatment with 1 µM FK506 or 10 µM CiP. These pretreatments did not affect amylase secretion or the rise in cytosolic Ca²⁺ after caerulein stimulation. The findings suggest that calcineurin mediates caerulein-induced intra-acinar zymogen activation, but not enzyme secretion nor the initial caerulein-induced cytosolic Ca²⁺ signal.