Abstract: Catechols are central intermediates in the metabolism of aromatic compounds. Degradation of 4-methyl-catechol via intradiol cleavage usually leads to the formation of 4-methylmuconolactone (4-ML) as a dead-end metabolite. Only a few microorganisms are known to mineralize 4-ML. The mml gene cluster of Pseudomonas reinekei MT1, which encodes enzymes involved in the metabolism of 4-ML, is shown here to encode 10 genes found in a 9.4-kb chromosomal region. Reverse transcription assays revealed that these genes form a single operon, where their expression is controlled by two promoters. Promoter fusion assays identified 4-methyl-3-oxoadipate as an inducer. Mineralization of 4-ML is initiated by the 4-methylmuconolactone methylisomerase encoded by mmlI. This reaction produces 3-ML and is followed by a rearrangement of the double bond catalyzed by the methylmuconolactone isomerase encoded by mmlJ. Deletion of mmlL, encoding a protein of the metallo-beta-lactamase superfamily, resulted in a loss of the capability of the strain MT1 to open the lactone ring, suggesting its function as a 4-methyl-3-oxoadipate enol-lactone hydrolase. Further metabolism can be assumed to occur by analogy with reactions known from the 3-oxoadipate pathway. mmlF and mmlG probably encode a 4-methyl-3-oxoadipyl-coenzyme A (CoA) transferase, and the mmlC gene product functions as a thiolase, transforming 4-methyl-3-oxoadipyl-CoA into methylsuccinyl-CoA and acetyl-CoA, as indicated by the accumulation of 4-methyl-3-oxoadipate in the respective deletion mutant. Accumulation of methylsuccinate by an mmlK deletion mutant indicates that the encoded acetyl-CoA hydrolase/transferase is crucial for channeling methylsuccinate into the central metabolism.