Methanogenesis, the biological production of methane, is the sole means for energy conservation for methanogenic archaea. Among the few methanogens shown to grow on carbon monoxide (CO) is Methanosarcina acetivorans, which produces, beside methane, acetate and formate in the process. Since CO-dependent methanogenesis proceeds via formation of formylmethanofuran from CO₂ and methanofuran, catalyzed by formylmethanofuran dehydrogenase, we were interested whether this activity could participate in the formate formation from CO. The genome of M. acetivorans encodes four putative formylmethanofuran dehydrogenases, two annotated as molybdenum-dependent and the remaining two as tungsten-dependent enzymes. A mutant lacking one of the putative tungsten enzymes grew very slowly on CO and only after a prolonged adaptation period, which suggests an important role for this isoform during growth on CO. Methanol- and CO-dependent growth of the mutant required the presence of molybdenum indicating an indispensable function of this metal in the remaining isoforms. CO-dependent formate formation could not be observed in the mutant indicating involvement of the respective isoform in the process. However, addition of formaldehyde, which spontaneously reacts with tetrahydrosarcinapterin (H₄SPT) to methenyl-H₄SPT, led to near-wild-type formate production rates, which argues for an alternative route of formate formation in this organism.