Premature intracellular activation of the digestive enzyme trypsinogen is considered to be the initiating event in pancreatitis. However, the direct consequences of intracellular trypsin activity have not previously been examined. In the current study, a mutant trypsinogen (paired basic amino acid cleaving enzyme (PACE)-trypsinogen), which is activated intracellularly by the endogenous protease PACE, was developed. This new construct allowed for the first time direct examination of the effects of intracellular trypsin on pancreatic acinar cells. We found that PACE-trypsinogen was expressed in the secretory pathway and was activated within acinar cells. Expression of PACE-trypsinogen induced apoptosis of HEK293 cells and pancreatic acinar cells, as indicated by histology, DNA laddering, PARP cleavage, and caspase-3 activation. Cell death was blocked by the trypsin inhibitor Pefabloc but not by the pancaspase inhibitor benzyloxycarbonyl-VAD, indicating that caspase-independent pathways were also involved. However, intracellular trypsin had no significant effect on the activity of the proinflammatory transcription factor NF-kappaB. In contrast, extracellular trypsin caused cell damage and dramatically increased NF-kappaB activity. These data indicate that localization of active trypsin determines its effects on pancreatic acinar cells. This new model will greatly improve our understanding of the role of active trypsin in pancreatitis and its associated inflammatory response.