The objective of this work is to investigate the area around the active sites of enzymes by the use of bifunctional irreversible inhibitors, so designed that they become chemically or physically bound to the active site, and then, already fixed in position, react with another amino acid residue in the vicinity. An inhibitor of this type, which becomes chemically bound to the active site of chymotrypsin, has been found to modify a methionine residue near it. The same methionine residue is also modified by a series of alkylating agents which are physically bound to the active site by virtue of a benzene ring, which they all contain. Inhibitors of the latter type have been used to label a serine residue at the active site of trypsin and a tyrosine residue at the active site of carboxypeptidase B. The active site of chymotrypsin has also been investigated with substrates of restricted conformation in order to determine the conformation of typical substrates as they interact with the active site. Work of a like nature is under way with human thrombin using peptide substrates and reversible and irreversible inhibitors derived from them in order to map the active site, find out why thrombin is so much more specific than trypsin, and possibly develop thrombin inhibitors for clinical use.