Abstract A series of N-alkylmaleimides was shown to effectively inhibit d-amino acid oxidase at pH 7.0 and 7.5. Apparent second order rate constants for maleimide inactivation of the enzyme increased with increasing chain length of the alkyl substituents of the maleimide derivatives. It is suggested that this chain length effect is due to an enhanced binding of the longer chain compounds through nonpolar interactions with the enzyme. N-Phenylmaleimide and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide were used to label d-amino acid oxidase. Product studies indicated that the maleimides were reacting only with cysteine residues of the enzyme. Peptide mapping was used to show that d-amino acid oxidase is composed of identical subunits having a molecular weight of 50,000. Product studies and spectrophotometric studies indicated that 3 moles of maleimide were bound per 50,000 g of protein. d-Amino acid oxidase was shown to be saturated with high concentrations of the N-alkylmaleimides, thus indicating the binding of the maleimides to the enzyme before the rate-limiting inactivation step. FAD and adenine derivatives were very effective in protecting the enzyme against inactivation by N-ethylmaleimide. Prior inactivation of the enzyme by N-ethylmaleimide prevented the binding of FAD to the enzyme. These results indicated that there is at least one reactive sulfhydryl group made less accessible by the binding of adenine derivatives at the FAD-binding site of d-amino acid oxidase.