We have isolated and identified a new zymogen in human pancreatic tissue and fluid. It is secreted as a minor component of pancreatic juice and resembles the two known trypsinogen variants in many properties. Its electrophoretic mobility and isoelectric pH lie between those of the cationic and anionic trypsinogen variants, and we propose the name "mesotrypsinogen" for the new enzyme precursor. It is activated by enteropeptidase or trypsin, and the free enzyme possesses a substrate specificity similar to that of the trypsins. Its pH optimum is at 8.2, and it appears to require Ca2+ for full enzymatic activity. The molecular weight of the new enzyme is approximately 25,000, similar to that of the known trypsin variants. Its stability resembles that of anionic trypsin extending over a pH range of 4-8.5. Activity is lost gradually at pH 2. The enzyme is inactivated rapidly by diisopropylfluorophosphate, but in contrast to the trypsins, it reacts only slowly with tosyllysine chloromethylketone. Immunologically, it is different from the cationic trypsin variant with which it does not cross-react. The most remarkable property of mesotrypsin is its almost total resistance to biological trypsin inhibitors, such as pancreatic trypsin inhibitor, soybean, lima bean, ovomucoid inhibitor, alpha 1-antitrypsin, etc. It is capable of activating trypsinogen in the presence of excess pancreatic trypsin inhibitor and thus inducing activation of other pancreatic zymogens, but it also possesses the ability to degrade trypsinogen rapidly to inert products. The physiological or pathophysiological role of this unique enzyme remains to be explored.