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The mbd cluster encoding genes of the 3‐methylbenzoyl‐CoA pathway involved in the anaerobic catabolism of 3‐methylbenzoate and m‐xylene was characterized for the first time in the denitrifying β‐Proteobacterium Azoarcus sp. CIB. The mbdA gene product was identified as a 3‐methylbenzoate‐CoA ligase required for 3‐methylbenzoate activation; its substrate spectrum was unique in activating all three methylbenzoate isomers. An inducible 3‐methylbenzoyl‐CoA reductase (mbdONQP gene products), displaying significant amino acid sequence similarities to known class I benzoyl‐CoA reductases catalysed the ATP‐dependent reduction of 3‐methylbenzoyl‐CoA to a methyldienoyl‐CoA. The mbdW gene encodes a methyldienoyl‐CoA hydratase that hydrated the methyldienoyl‐CoA to a methyl‐6‐hydroxymonoenoyl‐CoA compound. The mbd cluster also contains the genes predicted to be involved in the …