The first intermediate of anaerobic toluene catabolism, (R)-benzylsuccinate, is formed by enzymic addition of the methyl group of toluene to a fumarate cosubstrate and is subsequently activated to (R)-2-benzylsuccinyl-CoA. This compound is then oxidised to benzoyl-CoA and succinyl-CoA by a specific beta-oxidation pathway. The enzyme catalysing the first oxidation step of this pathway, (R)-benzylsuccinyl-CoA dehydrogenase, is encoded by the bbsG gene in Thauera aromatica. It was functionally overproduced in Escherichia coli, purified and characterised. The enzyme is a homotetramer with a subunit size of 45 kDa and contains one FAD per subunit. It is highly specific for (R)-benzylsuccinyl-CoA and is inhibited by (S)-benzylsuccinyl-CoA. An apparent K(m) value of 110+/-10 micro M was obtained for (R)-benzylsuccinyl-CoA. The reaction product of (R)-benzylsuccinyl-CoA dehydrogenase was identified as (E)-benzylidene-succinyl-CoA by comparison with the chemically synthesised compound, which was obtained via a new synthetic procedure. (R)-Benzylsuccinyl-CoA dehydrogenase was detected as a specifically substrate-induced protein in toluene- and m-xylene-grown cells of several bacterial species, using enzyme activity and immunological detection.