The membrane fraction isolated from broken cells of Methanobacterium ruminantium actively synthesized methane from CO2 and H2 without the addition of ATP or other cofactors. This activity was lost unless strictly anaerobic conditions were maintained throughout the isolation and incubation procedures. 3H2, but not 3H2O, was readily incorporated into methane. This indicates that hydrogen atoms are used in the formation of methane without the prior equilibration of protons with the water phase. Methylenetetrahydrofolate was shown to be converted into methane, but less efficiently than CO2. The evidence indicates that tetrahydrofolate derivatives may not be of primary importance in the formation of methane from CO2 and H2. No requirement for ATP in methanogenesis could be demonstrated. However, chemical reagents that can increase proton conductance in membranes and therby abolish the membrane electrical potential were also effective inhibitors of methanogenesis. It was postulated that, although the reduction of CO2 to methane by bacterial membranes may require energy derived from a transmembrane potential, this does not appear to be coupled to the intermediary synthesis of ATP.