Cell suspensions of Methanosarcina barkeri were found to oxidize formaldehyde to CO2 and 2H2 (delta G0' = -27 kJ/mol CO2), when methanogenesis was inhibited by 2-bromoethanesulfonate. We report here that this reaction is coupled with (a) primary electrogenic Na+ translocation at a stoichiometry of 2-3 Na+/CO2, (b) with secondary H+ translocation via a Na+/H+ antiporter and (c) with ATP synthesis driven by an electrochemical proton potential. This is concluded from the following findings. Formaldehyde oxidation to CO2 and 2H2 was dependent on Na+ ions, 2-3 mol Na+/mol formaldehyde oxidized were extruded. Na+ translocation was inhibited by Na+ ionophores, but not affected by protonophores of Na+/H+ antiport inhibitors. Formaldehyde oxidation was associated with the build up of a membrane potential in the order of 100 mV (inside negative), which could be dissipated by sodium ionophores rather than by protonophores. Formaldehyde oxidation was coupled with ATP synthesis, which could be inhibited by Na+ ionophores, Na+/H+ antiport inhibitors, by protonophores and by the H+-translocating-ATP-synthase inhibitor, dicyclohexylcarbodiimide. With cell suspensions of Methanobacterium thermoautotrophicum similar results were obtained.