Cell suspensions of Methanobacterium thermoautotrophicum were found to reduce CO2 with H2 to CO at a maximal rate of 100 nmol X min-1 X mg protein-1. Half-maximal rates were obtained at a H2 and a CO2 concentration in the gas phase of 10% and 30%, respectively. The CO concentration in the gas phase surpassed the equilibrium concentration by a factor of more than 15 which indicates that CO2 reduction with H2 to CO was energy-driven. This was substantiated by the observation that the cells only formed CO when they also generated methane and that CO formation was completely inhibited by uncouplers. CO formation by cell suspensions and by growing cells was inhibited by cyanide. Neither methane formation nor the electrochemical proton potential were affected by this inhibitor. Cyanide was shown to inactivate specifically the carbon monoxide dehydrogenase present in M. thermoautotrophicum. It is therefore concluded that reduction of CO2 to CO is catalyzed by this enzyme. CO production by growing cells was 5-10-times slower than by resting cells. This is explained by effective CO assimilation in growing cells; when CO assimilation was inhibited by propyl iodide the rate of CO production immediately increased more than tenfold.