D-amino acid oxidase from the yeast Rhodotorula gracilis is irreversibly inactivated by reaction with TNBS with complete inactivation accompanied by covalent modification of lysine residues of the protein. The inactivation was biphasic, the fast phase being dependent on TNBS concentration and completed in less than 1 minute. The competitive inhibitor benzoate afforded partial protection against inactivation during the fast phase of the process, with no effect on the slow phase. The pH curve of inactivation (slow phase) indicates the involvement of a residue(s) with a pK of 8.2. Amino acid analyses showed that in the fast phase of inactivation 1.6 lysine residues were modified, whereas up to 13 lysine residues were modified in the slow phase of inactivation. Our data show that TNBS behaves as an active site-directed reagent in yeast D-amino acid oxidase and they suggest the presence of at least one essential lysyl residue at or near the active site.