The flavoenzyme D-amino acid oxidase (Eo) is rapidly chlorinated by N-chloro-D-leucine (Rudie, N.G., Porter, D.J.T., and Bright, H.J. (1980) J. Biol. Chem. 255, 498-508). We have carried out chymotryptic digestion of E0-36Cl2 and find that all of the radiolabel is located in a heptapeptide having [3.5-36Cl2]chlorotyrosine as the COOH-terminal residue. This heptapeptide, having the sequence -Asp-Leu-Glu-Arg-Gly-Ile-Tyr-, is located within a larger fragment obtained previously from cyanogen bromide cleavage of E0. These results demonstrate that the target for chlorination in E0 must be a single tyrosine residue and provide, when taken together with previous findings, the first clear evidence for the identity and location of an active site residue in the polypeptide chain of D-amino oxidase.