Archaeoglobus fulgidus, a hyperthermophilic sulfate-reducing archaeon, was found to contain a membrane-bound F42JI2 : quinone oxidoreductase complex presumed to be involved in energy conservation during growth on lactate plus sulfate. After solubilization with dodecyl-/I-D-maltoside the complex was purified 32-fold with a yield of 24%. Using both gel filtration and native PAGE, an apparent molecular mass of approximately 270 kDa was determined. SDSPAGE revealed the presence of at least seven polypeptides with apparent molecular masses 56, 45, 41, 39, 37, 33, and 32 kDa. The purified complex contained 1.6 mol FAD, 9 mol non-heme iron and 7 mol acid-labile sulfur/mol complex. It did not contain cytochromes, which were, however, present in the membrane fraction of A. fulgidus (3 nmoVmg membrane protein). The purified F42& : quinone oxidoreductase complex catalyzed the reduction of 2,3-dimethyl-l,4-naphthoquinone (apparent K, 190 pM) with reduced coenzyme F420 (apparent K, 50 pM) exhibiting a specific activity of 500 U/mg (apparent VmJ at pH 8.0 (pH optimum) and 65 "C (temperature optimum). 2-Methyl-l,4-naphthoquinone (menadione), 2-hydroxy-l,4-naphthoquinone, 1,4-naphthoquinone, 2,3-dimethoxy-5-methyl-l,4-benzoquinone, and 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (decyl-ubiquinone) were also reduced with F,,,H,, albeit with lower rates. The physiological electron acceptor of the F4,aZ : quinone oxidoreductase complex is most likely the menaquinone found in the membrane fraction of A. fulgidus.