Coenzyme F420 is the central low-redox-potential electron carrier in methanogenic metabolism. The coenzyme is reduced under hydrogen by the action of F420-dependent hydrogenase. The standard free-energy change at pH 7 of F420 reduction was determined to be -15 kJ mol(-1), irrespective of the temperature (25-65 degrees C). Experiments performed with methane-forming cell suspensions of Methanothermobacter thermautotrophicus incubated under various conditions demonstrated that the ratios of reduced and oxidized F420 were in thermodynamic equilibrium with the gas-phase hydrogen partial pressures. During growth in a fed-batch fermenter, ratios changed in connection with the decrease in dissolved hydrogen. For most of the time, the changes were as expected for thermodynamic equilibrium between the oxidation state of F420 inside the cells and extracellular hydrogen. Also, methanol-metabolizing, but not acetate-converting, cells of Methanosarcina barkeri maintained the ratios of reduced and oxidized coenzyme F420 in thermodynamic equilibrium with external hydrogen. The results of the study demonstrate that F420 is a useful probe to assess in situ hydrogen concentrations in H2-metabolizing methanogens.