Protease inhibitors from the Kunitz family are widely distributed in plant kingdom. They share a conserved b-trefoil fold in which variable loops are involved in interactions with proteases. The majority of the Kunitz inhibitors are targeting serine proteases using canonical (Laskowski) mechanism based on a single binding loop with conserved structure. Here, we present high-resolution crystal structures of two potato Kunitz inhibitors in complexes with the serine protease trypsin. We identified a new, non-canonical type of a reactive center on these inhibitors that is formed by two loops. Furthermore, we employed cross-linking mass spectrometry approach to demonstrate that the non-canonical reactive site interacts also with chymotrypsin.