The two human anionic trypsinogens 1 and 2 were purified from human pancreatic juice by gel filtration on Sephadex G-100 and by chromatography on DEAE-cellulose. After activation of their respective zymogens by porcine enterokinase, human trypsins 1 and 2 were studied for their reaction with a wide variety of proteinase inhibitors. Kunitz pancreatic trypsin inhibitor and human pancreatic secretory trypsin inhibitor completely inhibited both human trypsins at a stoichiometric inhibitor-to-enzyme ratio of one to one. In contrast, bovine pancreatic secretory trypsin inhibitor (Kazal's inhibitor) failed to inhibit either human trypsin. The inhibition of both human trypsins by porcine pancreatic secretory trypsin inhibitor was demonstrated. The reactions of the trypsins with chicken ovomucoid, Ascaris lumbricoides (type suis), human sperm and blood plasma trypsin inhibitors were studied. The most striking difference between the two human trypsins was the reaction with soybean trypsin inhibitor (Kunitz). Trypsin 2 was completely inhibited in a one-to-one molar ratio while trypsin 1 was poorly inhibited. The presence of a prekallikrein in human pancreatic juice is discussed.