The synthesis of formyl-methanofuran and the reduction of the heterodisulfide (CoM-S-S-CoB) of coenzyme M (HS-CoM) and coenzyme B (HS-CoB) are two crucial, H 2 -dependent reactions in the energy metabolism of methanogenic archaea. The bioenergetics of the reactions in vivo were studied in chemostat cultures and in cell suspensions of Methanothermobacter thermautotrophicus metabolizing at defined dissolved hydrogen partial pressures ( p H 2 ). Formylmethanofuran synthesis is an endergonic reaction (DG°¢ ¼ +16 kJAEmol )1 ). By analyzing the concentration ratios between formyl-methanofuran and methanofuran in the cells, free energy changes under experimental conditions (DG¢) were found to range between +10 and +35 kJAEmol )1 depending on the p H 2 applied. The comparison with the sodium motive force indicated that the reaction should be driven by the import of a variable number of two to four sodium ions.Heterodisulfide reduction (DG°¢ ¼ )40 kJAEmol )1 ) was associated with free energy changes as high as )55 to )80 kJAEmol )1 . The values were determined by analyzing the concentrations of CoM-S-S-CoB, HS-CoM and HS-CoB in methane-forming cells operating under a variety of hydrogen partial pressures. Free energy changes were in equilibrium with the proton motive force to the extent that three to four protons could be translocated out of the cells per reaction. Remarkably, an apparent proton translocation stoichiometry of three held for cells that had been grown at p H 2 <0.12 bar, whilst the number was four for cells grown above that concentration. The shift occurred within a narrow p H 2 span around 0.12 bar. The findings suggest that the methanogens regulate the bioenergetic machinery involved in CoM-S-S-CoB reduction and proton pumping in response to the environmental hydrogen concentrations.Keywords: energy conservation; methanogenesis; proton motive force; sodium motive force; Methanothermobacter thermautotrophicus.Methanothermobacter thermautotrophicus is a methanogenic Archaeon that derives the energy for autrophic growth from the reduction of CO 2 with molecular hydrogen as the electron donor. The process of methanogenesis consists of a series of reduction reactions at which the one-carbon unit derived from CO 2 is bound to C 1 carriers of unique nature (for recent reviews see [1,2]). From a bioenergetic point of view, three reactions are of importance, notably the formation of formyl-methanofuran, the N 5 -methyl-tetrahydromethanopterin:coenzyme M methyl transfer step and the H 2 -dependent reduction of CoM-S-S-CoB [1,[3][4][5].Formyl-methanofuran (MFR-NH-CHO; f-MFR) synthesis represents the first step in methanogenesis. In this step, CO 2 is bound to methanofuran (MFR-NH 3 + ; MFR) and subsequently reduced to the formyl state with electrons derived from hydrogen (reaction 1). MFR-NHThe reaction is endergonic under thermodynamic standard conditions [1,6]. Studies with cell suspensions of Methanosarcina barkeri and Methanothermobacter marburgensis indicated that reaction (1) is driven by a sodium motive