The mechanism of action of trypsin and other serine proteases has been widely debated, particularly with regard to the identification of the group at the active site which functions as the chemical base during the catalytic process. Attempts to resolve this question by a number of indirect techniques, including NMR, isotope exchange, difference IR and quantum mechanical calculations, have resulted in different identifications of this group. Neutron diffraction, because of its ability to locate hydrogen atoms experimentally, offers the most direct way of resolving this issue. Results are presented here from a 2.2-A neutron data set for bovine trypsin covalently inhibited by a transition-state analogue, the monoisopropylphosphoryl (MIP) group. His 57 is clearly identified as the base in the catalytic process.