Chemical modification of a single tryptophan residue in antithrombin III with dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide blocks heparin binding and the heparin-enhanced inhibition of thrombin without altering the heparin-independent rate of thrombin inhibition (Blackburn, M. N., and Sibley, C. C. (1980) J. Biol. Chem. 255, 824-826). The labeled protein was reduced and carboxymethylated and then cleaved with cyanogen bromide. The peptide containing the hydroxynitrobenzyl-labeled tryptophan was isolated by gel filtration and ion exchange chromatography. Amino acid analysis of the labeled peptide indicates that it corresponds to residues 21 through 89 of human antithrombin III. The site of labeling corresponds to Trp 49, which is located within the disulfide-stabilized loops near the NH2-terminal end of the antithrombin III molecule.