D-Amino-acid oxidase from Rhodotorula gracilis was irreversibly inactivated by phenylglyoxal in a biphasic process. The fast phase was completed in less than 1 min. Its extent was linearly dependent on phenylglyoxal concentration and was not influenced by the presence of FAD or benzoate, a pseudo-substrate. The second phase of inactivation was due to a simple second-order reaction. The presence of FAD exerted only partial protection; the second-order rate constants of inactivation were 8.3 M-1 min-1 for holoprotein and 18.0 M-1 min-1 for apoprotein. The addition of benzoate completely protected against this second phase of inactivation. Efforts to isolate the enzyme modified at a single arginine residue at the end of the fast phase were unsuccessful, but analysis of the enzyme isolated at the end of the slow phase identified an arginine residue, protected by benzoate, that is highly conserved in all D-amino-acid oxidases and corresponds to Arg283 in the pig kidney enzyme. Modification of this residue is directly involved in the inactivation process during the slow phase. This arginine may represent the basic residue ion pairing with the carboxylate group of the substrate or the residue interacting with the flavin N1-C2 = O locus.