All methanogenic Archaea examined to date rely on methanogenesis as their sole means of energy conservation. Among these are ones that use carbon monoxide as a growth substrate, producing methane via a pathway that involves hydrogen as an intermediate. To further examine the role of hydrogen in this process, we tested the ability of Methanosarcina acetivorans C2A, a metabolically versatile methanogen devoid of significant hydrogen metabolism, to use CO as a growth substrate. M. acetivorans grew on CO to high cell densities (Ϸ1 ؋ 10 8 per ml) with a doubling time of Ϸ24 h. Surprisingly, acetate and formate, rather than methane, were the major metabolic end products as shown by 13 C NMR studies and enzymatic analysis of culture supernatants. Methane formation surpassed acetate͞formate formation only when the cultures entered stationary growth phase, strongly suggesting that M. acetivorans conserves energy by means of this acetogenic and formigenic process. Resting cell experiments showed that methane production decreased linearly with increasing CO partial pressures, consistent with inhibition of methanogenesis by CO. Transposoninduced M. acetivorans mutants with lesions in the operon encoding phosphotransacetylase and acetate kinase failed to use either acetate or CO as growth substrates, indicating that these enzymes are required for both aceticlastic methanogenesis and carboxidotrophic acetogenesis. These findings greatly extend our concept of energy conservation and metabolic versatility in the methanogenic Archaea.acetogenesis ͉ methanogenesis T he only known pathway for energy conservation in methanogenic Archaea is methanogenesis. In these organisms, methane is produced either by the stepwise reduction of CO 2 via cofactor-bound intermediates or by transfer of methyl groups from methylated compounds to a coenzyme and subsequent reduction to methane (reviewed in refs. 1-3). Although most methanogens are able to reduce CO 2 by using H 2 as a reductant, only members of the Methanosarcinales use acetate and methylated compounds, such as methanol or methylamines, for growth as well. These compounds serve as both electron donors and acceptors for the methanogenic process. Also, two methanogenic species have been shown to use carbon monoxide (CO) as a methanogenic growth substrate, whereas Methanosphaera species are able to grow on the combination of methanol and H 2 . Regardless of the substrate, methane and CO 2 are the only major products of all methanogenic bioconversions; although some other products occasionally have been detected, these are generated only in minor amounts (4-8). Thus, all methanogens examined to date are obligate methanogens.Microbial CO consumption is an environmentally important process that fuels the reentry of CO into the global carbon cycle and helps maintain atmospheric CO below toxic levels (9). CO oxidation is a property of numerous bacterial genera, both aerobic and anaerobic. Phototrophic anaerobes such as Rhodocyclus gelatinosus and Rhodospirillum rubrum couple CO oxidation, whic...