Catechol 1,2-dioxygenase I₁ (CDI₁) is the first enzyme of the β-ketoadipate pathway in Acinetahacter lowffii K24. CDI₁ has two cysteines (155, 202) and its enzyme activity is inhibited by the cysteine inhibitor, AgNO₃. Two mutants, CDI₁ C155V and CDI₁ C202V, were obtained by sitedirected mutagenesis. The two mutants were overexpressed and the mutated amino acid residues (Cys→Val) were characterized by pegtide mapping and amino acid sequencing. Interestingly, CDI₁ C155V was inhibited by AgNO₃, whereas CDI₁ C202V was not inhibited. This suggests that Cys^(202) is the sole inhibition site by AgNO₃ and is close to the active site of the enzyme. However, the results of the biochemical assay of mutated CDI₁s suggest that the two cysteines are not directly involved in the activity of the catechol 1,2-dioxygenase of CDI₁.