An iron-sulfur flavoprotein (Isf) from the methanoarchaeaon Methanosarcina thermophila, which participates in electron transfer reactions required for the fermentation of acetate to methane, was characterized by electrochemistry and EPR and Mössbauer spectroscopy. The midpoint potential (Em) of the FMN/FMNH2 couple was -0.277 V. No flavin semiquinone was observed during potentiometric titrations; however, low amounts of the radical were observed when Isf was quickly frozen after reaction with CO and the CO dehydrogenase/acetyl-CoA synthase complex from M. thermophila. Isf contained a [4Fe-4S]2+/1+ cluster with g values of 2.06 and 1.93 and an unusual split signal with g values at 1.86 and 1.82. The unusual morphology was attributed to microheterogeneity among Isf molecules. The Em value for the 2+/1+ redox couple of the cluster was -0.394 V. Extracts from H2-CO2-grown Methanobacterium thermoautotrophicum cells catalyzed either the H2- or CO-dependent reduction of M. thermophila Isf. In addition, Isf homologs were found in the genomic sequences of the CO2-reducing methanoarchaea M. thermoautotrophicum and Methanococcus jannaschii. These results support a general role for Isf in electron transfer reactions of both acetate-fermenting and CO2-reducing methanoarchaea. It is suggested that Isf functions to couple electron transfer from ferredoxin to membrane-bound electron carriers, such as methanophenazine and/or b-type cytochromes.