Pyrocatechase (catechol 1,2-oxidoreductase (decyclizing), EC 1.13.11.1), a ferric ion-containing dioxygenase from Pseudomonas arvilla C-1, catalyzes the intradiol cleavage of catechol with insertion of 2 atoms of molecular oxygen to form cis,cis-muconic acid. The enzyme also catalyzed the oxidation of various catechol derivatives, including 4-methylcatechol, 4-chlorocatechol, 4-formylcatechol (protocatechualdehyde), 4,5-dichlorocatechol, 3,5-dichlorocatechol, 3-methylcatechol, 3-methoxycatechol, and 3-hydroxycatechol (pyrogallol). All of these substrates gave products having an absorption maximum at around 260 nm, which is characteristic of cis,cis-muconic acid derivatives. However, when 3-methylcatechol was used as substrate, the product formed showed two absorption maxima at 390 and 260 nm. These two absorption maxima were found to be attributable to two different products, 2-hydroxy-6-oxo-2,4-heptadienoic acid and 5-carboxy-2-methyl-2,4-pentadienoic acid (2-methylmuconic acid). The former was produced by the extradiol cleavage between the carbon atom carrying the hydroxyl group and the carbon atom carrying the hydroxyl group and the carbon atom carrying the methyl group; the latter by an intradiol cleavage between two hydroxyl groups. Since these products were unstable, they were converted to and identified as 6-methylpyridine-2-carboxylic acid and 2-methylmuconic acid dimethylester, respectively. Similarly, 3-methoxycatechol gave two products, namely, 2-hydroxy-5-methoxycarbonyl-2,4-pentadienoic acid and 5-carboxy-2-methoxy-2,4-pentadienoic acid (2-methoxymuconic acid). With 3-methylcatechol as substrate, the ratio of intradiol and extradiol cleavage activities of Pseudomonas pyrocatechase during purification was almost constant and was about 17. The final preparation of the enzyme was homogeneous when examined by disc gel electrophoresis and catalyzed both reactions simultaneously with the same ratio as during purification. All attempts to resolve the enzyme into two components with separate activities, including inactivation of the enzyme with urea or heat, treatment with sulfhydryl-blocking reagents or chelating agents, and inhibition of the enzyme with various inhibitors, proved unsuccessful. These results strongly suggest that Pseudomonas pyrocatechase is a single enzyme, which catalyzes simultaneously both intradiol and extradiol cleavages of some 3-substituted catechols.