Most methanogenic Archaea contain an unusual cytoplasmic fumarate reductase which catalyzes the reduction of fumarate with coenzyme M (CoM-S-H) and coenzyme B (CoB-S-H) as electron donors forming succinate and CoM-S-S-CoB as products. We report here on the purification and characterization of this thiol:fumarate reductase (Tfr) from Methanobacterium thermoautotrophicum (strain Marburg). The purified enzyme, which was composed of two different subunits with apparent molecular masses of 58 kDa (TfrA) and 50 kDa (TfrB), was found to catalyze the following reactions: (a) the reduction of fumarate with CoM-S-H and CoB-S-H (150 U/mg); (b) the reduction of fumarate with reduced benzyl viologen (620 U/mg); (c) the oxidation of CoM-S-H and CoB-S-H to CoM-S-S-CoB with methylene blue (95 U/mg); and (d) the reduction of CoM-S-S-CoB with reduced benzyl viologen (250 U/mg). The flavoprotein contained 12 mol non-heme iron and approximately the same amount of acid-labile sulfur/mol heterodimer. The genes encoding TfrA and TfrB were cloned and sequenced. Sequence comparisons with fumarate reductases and succinate dehydrogenases from Bacteria and Eucarya and with heterodisulfide reductases from M. thermoautotrophicum and Methanosarcina barkeri revealed that TfrA harbors FAD-binding motifs and the catalytic site for fumarate reduction and that TfrB harbors one [2Fe-2S] cluster and two [4Fe-4S] clusters and the catalytic site for CoM-S-H and CoB-S-H oxidation.