Biochemical studies have revealed two distinct classes of Coenzyme B-Coenzyme M heterodisulfide (CoB-S-S-CoM) reductase (Hdr), a key enzyme required for anaerobic respiration in methane-producing archaea. A cytoplasmic HdrABC enzyme complex is found in most methanogens, whereas a membrane-bound HdrED complex is found exclusively in members of the order Methanosarcinales. Unexpectedly, genomic data indicate that multiple copies of both Hdr classes are found in all sequenced Methanosarcinales genomes. The Methanosarcina acetivorans hdrED1 operon is constitutively expressed and required for viability under all growth conditions examined, consistent with HdrED being the primary Hdr. HdrABC appears to be specifically involved in methylotrophic methanogenesis, based on reduced growth and methanogenesis rates of an hdrA1C1B1 mutant on methylotrophic substrates and downregulation of the genes during growth on acetate. This conclusion is further supported by phylogenetic analysis showing that the presence of hdrA1 in an organism is specifically correlated with the presence of genes for methylotrophic methanogenesis. Examination of mRNA abundance in methanol-grown Delta hdrA1C1B1 strains relative to wild-type revealed upregulation of genes required for synthesis of (di)methylsulfide and for transport and biosynthesis of CoB-SH and CoM-SH, suggesting that the mutant has a defect in electron transfer from ferredoxin to CoB-S-S-CoM that causes cofactor limitation.