The inactivation of chymotrypsin A4 and B by the bifunctional reagents L-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone (L-TPCK) and phenoxymethyl chloromethyl ketone (PMCK) has been investigated. The rate of inactivation of chymotrypsin A4 with both reagents as a function of pH has been shown to be dependent on a basic group of pK = 6.3–6.5. Chymotrypsin B inactivation appears to be dependent on a basic group with a somewhat lower pK. For each enzyme the reaction with both reagents is associated with the loss of a single histidine residue. By the isolation and identification of 3-carboxymethylhistidine from the alkylated and oxidized peptic histidine-57 peptides, it has been concluded that both enzymes are alkylated at the nitrogen-3 position of histidine-57 by L-TPCK and PMCK. Evidence for the submolar alkylation of methionine-192 of chymotrypsin A4 by L-TPCK, PMCK, D-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone (D-TPCK), and N-methyl-L-TPCK is presented. There is no alkylation of the histidine of chymotrypsin A4 by D-TPCK or.N-methyl-L-TPCK.From a comparison of the structures of a number of reagents known to alkylate chymotrypsin A4, it has been concluded that the alkylation of methionine-192 is nonspecific and relatively independent of any defined stereochemistry of the reagent employed. To date the alkylation of histidine-57 has been shown to occur only with haloketones, and is dependent on the distance between the haloketone and the aromatic ring when the latter is present. Although the presence of an asymmetric α-carbon and acylamido group in straight-chain reagents is unnecessary for histidine alkylation, these must be of the L configuration if present.