The obligately anaerobic, denitrifying bacteriumAzoarcus anaerobiusstrain LuFRes1 grows with resorcinol (1,3-dihydroxybenzene) as sole carbon and energy source. Resorcinol is oxidized to hydroxyhydroquinone (1,2,4-trihydroxybenzene) by resorcinol hydroxylase (RH), an inducible membrane-bound enzyme. Sequence comparison places resorcinol hydroxylase into the group of anaerobic molybdopterin oxidoreductases and dimethyl sulfoxide reductase-like enzymes. In the large subunit, a molybdopterin-binding domain was predicted, and the small subunit most likely contains two [4Fe-4S] centers. Growth of molybdate-starved cells was inhibited by tungstate, and in vitro resorcinol hydroxylase activity was inhibited by arsenite and selenite that are known to inhibit molybdenum-containing enzymes. The two genes encoding resorcinol hydroxylase could be expressed inEscherichia colibut the products remained in inclusion bodies.All attempts to purify RH fromA. anaerobiusor to produce soluble, active RH inE. colifailed. Nevertheless, RH was produced as a C-terminally Strep-tagged protein from plasmid pSKM1 inThauera aromaticaAR1 transconjugants carrying a transposon insertion in the coding gene for the large (Delta rhL) or the small subunit (Delta rhS) of RH from cosmid R+. RH in the membrane fraction of wild-type transconjugantT. aromaticaAR1/R(+)showed a specific activity of 80 mU mg(-1), and the specific activity of RH in the membranes of the complemented mutants was in the same range (80-95 mU mg(-1)). We conclude that RH ofA. anaerobiusis a membrane-bound molybdoenzyme consisting of two subunits which might require a further loosely bound subunit as membrane anchor.