Conversion of biomass-derived syngas (gaseous mixture of mainly H2, CO and CO2) to methane might be a sustainable alternative for the biofuel industry. Via the syngas route more methane can be produced from biomass than via conventional anaerobic digestion. Methanogenic archaea are key players in syngas conversion, but only a few are known to utilize CO (or syngas). Methanothermobacter thermoautotrophicus is one of the few hydrogenotrophic methanogens which has been observed to grow on CO. However, carboxydotrophic growth is slow and is reported to be readily inhibited above 50 kPa CO. The aim of this work was to get more insight of the CO metabolism in hydrogenotrophic archaea and to assess the potential toxic effects of CO towards these microorganisms. Archaeal genomic databases were searched for putative homologues of the Methanothermobacter thermoautotrophicus CODH alpha subunit (containing the catalytic site): the highest scores were for the CODH subunits of Methamothermobacter marburgensis (93% identity) and Methanococcus maripaludis (71%). M. thermoautotrophicus and the other two potential carboxydotrophic strains were incubated with CO and CO + H2 as sole substrates. In addition to M. thermoautotrophicus, M. marburgensis was able to grow methanogenically on CO alone and on CO + H2. In contrast to M. thermoautotrophicus, M. marburgensis was not as strongly inhibited when grown in presence of CO alone and was able to adapt its metabolism, shifting its lag phase from ~500 to ~100 hours. It was observed for both strains that presence of hydrogen stimulates the carbon monoxide metabolism. To gain further insight, the proteome of M. marburgensis culture grown on H2 + CO2 and H2 + CO2 + CO were analysed. Cultures grown with H2 + CO showed relative higher abundance of enzymes involved in CODH/ACS associated reactions and reactions involved in redox metabolism. Overall, the data suggests the strong reducing capacity of CO inhibits the hydrogenotrophic methanogen, making growth on CO as a sole substrate difficult for these organisms.