Due to autodigestion the activity of dissolved trypsin successively decreases. Autolysis leads to proteolytic cleavages of some arginyl and lysyl peptide bonds of the trypsin structure. Three important autolysis sites have been reported for bovine trypsin: Lys61-Ser62, Arg117-Val118 and Lys145-Ser146. Out of these three sites only the first two exist in rat trypsin, an enzyme that has been the target of protein engineering for more than ten years. In this work Lys61 and Arg117 were replaced by Asn via site directed mutagenesis to transform the corresponding peptide bonds to trypsin resistant ones. Kinetic parameters of K61N, R117N and the double mutant K61N/R117N are practically identical with those of the wild-type enzyme. By contrast, the rate of autolysis of each singly-substituted species is substantially slower than with the parent trypsin. In particular, the double mutant shows dramatically increased stability against autolysis and decreased sensitivity to Ca2+. The process of autolysis has been followed by N-terminal sequence determination. We propose a model to explain why these two positions play a key role in autolysis and how Ca2+ can influence this process. In addition, our in vitro results strongly support the recently proposed model of human hereditary pancreatitis.