Essential amino acids involved in the catalytic mechanisms of cytosine deaminase from Aspergillus fumigatus IFO 5840 were determined by chemical modification studies. The enzyme was perfectly inhibited by N-bromosuccinimide, chloramines-T, pyridoxal-5’-phosphate, and p-chloromercuribenzoate. It was strongly inhibited by phenylmethyl sulfonyl fluoride, and weakly inhibited by phenylglyoxal. The inactivation of the enzyme activity by p-CMB was reversed by sulfhydryl reagents. Furthermore, activities inhibited by chloramines-T, pyridoxal-5’-phosphate, results, we speculate that tryptophan, methionine, lysine and cysteine residues are located in ornear the active center of the cytosine deaminase, while a serine is indirectly involved I the enzyme activity.