Structural rationalizations for differences in catalytic efficiency and stability between mesophilic and cold-adapted trypsins have been suggested from a detailed comparison of eight trypsin structures. Two trypsins, from Antarctic fish and Atlantic cod, have been constructed by homology modeling techniques and compared with six existing X-ray structures of both cold-adapted and mesophilic trypsins. The structural analysis focuses on the cold trypsin residue determinants found in a more extensive comparison of 27 trypsin sequences, and reveals a number of structural features unique to the cold-adapted trypsins. The increased substrate affinity of the psychrophilic trypsins is probably achieved by a lower electrostatic potential of the S1 binding pocket particularly arising from Glu221B, and from the lack of five hydrogen bonds adjacent to the catalytic triad. The reduced stability of the cold trypsins is expected to arise from reduced packing in two distinct core regions, fewer interdomain hydrogen bonds and from a destabilized C-terminal alpha-helix. The helices of the cold trypsins lack four hydrogen bonds and two salt-bridges, and they have poorer van der Waals packing interactions to the body of the molecule, compared to the mesophilic counterparts.