Biochemical studies in Azoarcus sp. strain T have demonstrated that anaerobic oxidation of both toluene and m-xylene is initiated by addition of the aromatic hydrocarbon to fumarate, forming benzylsuccinate and 3-methyl benzylsuccinate, respectively. Partially purified benzylsuccinate synthase was previously shown to catalyze both of these addition reactions. In this study, we identified and sequenced the genes encoding benzylsuccinate synthase from Azoarcus sp. strain T and examined the role of this enzyme in both anaerobic toluene and m-xylene mineralization. Based on reverse transcription-PCR experiments and transcriptional start site mapping, we found that the structural genes encoding benzylsuccinate synthase, bssCAB, together with two additional genes, bssD and bssE, were organized in an operon in the order bssDCABE. bssD is believed to encode an activating enzyme, similar in function to pyruvate formate-lyase activase. bssE shows homology to tutH from Thauera aromatica strain T1, whose function is currently unknown. A second operon that is upstream of bssDCABE and divergently transcribed contains two genes, tdiS and tdiR. The predicted amino acid sequences show similarity to sensor kinase and response regulator proteins of prokaryotic two-component regulatory systems. A chromosomal null bssA mutant was constructed (the bssA gene encodes the ␣-subunit of benzylsuccinate synthase). This bssA null mutant strain was unable to grow under denitrifying conditions on either toluene or m-xylene, while growth on benzoate was unaffected. The growth phenotype of the ⌬bssA mutant could be rescued by reintroducing bssA in trans. These results demonstrate that benzylsuccinate synthase catalyzes the first step in anaerobic mineralization of both toluene and m-xylene.