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PMP-D2 and HI, two peptides from Locusta migratoria, were shown to belong to the family of tight-binding protease inhibitors. However, they interact weakly with bovine trypsin (K-i around 100 nM) despite a trypsin-specific Arg at the primary specificity site P1. Here we demonstrate that they are potent inhibitors of midgut trypsins isolated from the same insect and of a fungal trypsin from Fusarium oxysporum (K-i less than or equal to 0.02 nM). Therefore, they display a selectivity not existing for the parent chymotrypsin inhibitor PMP-C. By NMR, we demonstrate that HI possesses a highly rigid structure similar to the crystal structure of a variant of PMP-D2 in complex with bovine alpha-chymotrypsin. The main difference with PMP-C is located in the region from residues 20 to 24 (positions P6-P10) that interacts with the loop containing Gly173 in chymotrypsin. The corresponding residue in mammalian trypsins is always a proline that may generate a steric clash with the inhibitor. The residues thought to confer selectivity were mutated with PMP-C as a model. The resulting analogue PMP-D2(K10W,P21A,W25A) loses some activity toward insect and fungal trypsins but is a more potent inhibitor of mammalian trypsins, corresponding to a decrease of selectivity. This work represents a first attempt in tuning the selectivity of natural peptidic serine protease inhibitors by mutating residues out of the reactive loop (P3-P’3).