Archaeoglobus fulgidus, a hyperthermophilic sulfate-reducing archaeon, was found to contain a membrane-bound F420H2: quinone oxidoreductase complex presumed to be involved in energy conservation during growth on lactate plus sulfate. After solubilization with dodecyl-beta-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. SDS/PAGE 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 nmol/mg membrane protein). The purified F420H2: quinone oxidoreductase complex catalyzed the reduction of 2,3-dimethyl-1,4-naphthoquinone (apparent Km 190 microM) with reduced coenzyme F420 (apparent Km 50 microM) exhibiting a specific activity of 500 U/mg (apparent Vmax) at pH 8.0 (pH optimum) and 65 degrees C (temperature optimum). 2-Methyl-1,4-naphthoquinone (menadione), 2-hydroxy-1,4-naphthoquinone, 1,4-naphthoquinone, 2,3-dimethoxy-5-methyl-1,4- benzoquinone, and 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone (decyl-ubiquinone) were also reduced with F420H2, albeit with lower rates. The physiological electron acceptor of the F420H2: quinone oxidoreductase complex is most likely the menaquinone found in the membrane fraction of A. fulgidus.