The structure of trypsin from the fungusFusarium oxysporumhas been refined at 1.55 Å resolution by restrained least‐squares minimization to anR‐factor of 14.4%. The data were recorded from a single‐crystal on the X31 beamline at EMBL, Hamburg, using a locally developed image‐plate scanner. The final model consists of 1557 protein atoms, 400 water molecules, one molecule of isopropanol and one monoisopropyl phosphoryl inhibitor group covalently bound to the catalytic Ser195. Comparison of the structure with bovine trypsin reveals significant differences in the active site and suggests a possible explanation for the difference in substrate specificity between the two enzymes. InF. oxysporumtrypsin the specificity pocket is larger than in bovine trypsin. This explains the preference ofF. oxysporumtrypsin for the bulkier arginine over lysine and the reverse preference in bovine trypsin. The binding cavity on the C‐terminal side of the substrate is more restricted inF. oxysporumtrypsin than in mammalian andStreptomyces griseustrypsins, which explains the relative inactivity ofF. oxysporumtrypsin towards peptide–pNA substrate analogues as an unfavourable steric interaction between the side of the binding cavity and thepara‐nitroanilino group of peptide–pNA. The observed restriction of the binding cavity does not lead to a reduced catalytic activity compared