Four trypsin-chymotrypsin inhibitors from Syrian local small lentils were selected to study the reasons for their different action against human and bovine proteinases. Chemical modification experiments, enzymatic modifications followed by carboxypeptidase degradation, and characterization of the inhibitor/enzyme complexes formed were performed. All four Lens culinaris inhibitors (LCI) contained arginine at the trypsin-reactive site, and tyrosine (LCI-1.7 and LCI-2.2), phenylalanine (LCI-3.3), or leucine (LCI-4.6) at the chymotrypsin-reactive site. The inhibition of more than one molecule of human chymotrypsin per molecule of inhibitor was caused by the additional and atypical binding at the trypsin-reactive site of all four inhibitors. The approximately 2.5-fold higher inhibition of human chymotrypsin compared to bovine chymotrypsin was the result of two effects, the additional binding of human chymotrypsin at the trypsin-reactive site and the low inhibition of bovine chymotrypsin. As a consequence, human enzyme preparations or suitable conversion factors should be used to evaluate the effect of such inhibitors in foods.