Catechol 2,3-dioxygenase (C23D; EC 1.13.1.2) was purified to homogeneity from a cell extract of Pseudomonas sp. AW-2 grown on aniline, and the purified C23D was characterized. The molecular mass estimated by gel filtration was 110 kDa. The enzyme dissociated into four identical subunits each with the molecular mass of 33 kDa. The enzyme had high activity for 3-methylcatechol as well as catechol, and differed from the enzyme from Pseudomonas putida mt-2, which carries the TOL plasmid, in optimal pH for catechol, extradiol cleavage activities for 3-methylcatechol and 4-methylcatechol, and immunochemical properties. The amino acid sequence deduced from a C23D gene, alnE, from Pseudomonas sp. AW-2 was 85.7% identical to that of 3-methylcatechol 2,3-dioxygenase from toluidine-assimilating Pseudomonas putida UCC22. AlnE was 44.1% identical to the C23D encoded by xylE in P. putida mt-2. Because XylE has low activity for 3-methylcatechol, these results suggest that the differences in substrate specificity for 3-methylcatechol among the C23Ds reflected their sequence similarity.