Methanosarcina mazei is a methanogenic archaeon that is able to thrive on various substrates and therefore contains a variety of redox-active proteins involved in both cytoplasmic and membrane-bound electron transport. The organism possesses a complex branched respiratory chain that has the ability to utilize different electron donors. In this study, two knockout mutants of the membrane-bound F420 dehydrogenase (ΔfpoF and ΔfpoA-O) were constructed and analyzed. They exhibited severe growth deficiencies with trimethylamine, but not with acetate, as substrates. In cell lysates of the fpo mutants, the F420:heterodisulfide oxidoreductase activity was strongly reduced, although soluble F420 hydrogenase was still present. This led to the conclusion that the predominant part of cellular oxidation of the reduced form of F420 (F420H2) in Ms. mazei is performed by F420 dehydrogenase. Enzyme assays of cytoplasmic fractions revealed that ferredoxin (Fd):F420 oxidoreductase activity was essentially absent in the ΔfpoF mutant. Subsequently, FpoF was produced in Escherichia coli and purified for further characterization. The purified FpoF protein catalyzed the Fd:F420 oxidoreductase reaction with high specificity (the KM for reduced Fd was 0.5 μm) but with low velocity (Vmax = 225 mU·mg−1) and was present in the Ms. mazei cytoplasm in considerable amounts. Consequently, soluble FpoF might participate in electron carrier equilibrium and facilitate survival of the Ms. mazeiΔech mutant that lacks the membrane-bound Fd-oxidizing Ech hydrogenase.