The organization of all genes required for the anaerobic conversion of toluene to benzoyl-CoA was investigated in denitrifying Azoarcus-like strain EbN1. All of these genes are clustered within 25.3 kb of contiguous DNA sequence, which includes only a few intervening sequences. The toluene-catabolic genes are organized in two apparent operons. One contains the genes ( bssCAB) for the three subunits of benzylsuccinate synthase, which initiates anaerobic toluene degradation by converting toluene to ( R)-benzylsuccinate. The BssCAB proteins of strain EbN1 are most similar to those of Thauera aromatica strain K172. The bssCAB genes are part of a larger putative operon ( bssDCABEFGH), which contains the gene bssD, encoding the activase for benzylsuccinate synthase, and four genes ( bssEFGH) encoding proteins of unknown function. RT-PCR experiments showing continuation of transcription over the three largest intergenic regions of the bss operon support the assumed structure. Moreover, BssG was identified as toluene-induced protein. Downstream of the bss genes, another large putative operon ( bbsA- H) was identified that contains all genes required for beta-oxidation of benzylsuccinate to benzoyl-CoA, e.g. bbsEF, encoding succinyl-CoA:( R)-benzylsuccinate CoA-transferase. Immediately upstream of the bss operon, genes for a two-component regulatory system were identified; their products may sense toluene and induce the expression of both catabolic operons. The order and sequences of the bss and bbs genes are highly similar among toluene-degrading denitrifiers. The bss and bbs genes of the Fe(III)-reducing Geobacter metallireducens display less sequence similarity and are organized differently. The genes between the bss and bbs operons and in the flanking regions differ between strain EbN1 and the other strains.