Methanosarcina mazei belongs to the group of aceticlastic methanogens and converts acetate into the potent greenhouse gases CO2 and CH4. The aceticlastic respiratory chain involved in methane formation comprises the three transmembrane proteins Ech hydrogenase, F420 nonreducing hydrogenase and heterodisulfide reductase. It has been shown that the latter two contribute to the proton motive force. The data presented here clearly demonstrate that Ech hydrogenase is also involved in energy conservation. ATP synthesis was observed in a cytoplasm-free vesicular system of Ms. mazei that was dependent on the oxidation of reduced ferredoxin and the formation of molecular hydrogen (as catalysed by Ech hydrogenase). Such an ATP formation was not observed in a Δech mutant strain. The protonophore 3,5-di-tert-butyl-4-hydroxybenzylidene-malononitrile (SF6847) led to complete inhibition of ATP formation in the Ms. mazei wild-type without inhibiting hydrogen production by Ech hydrogenase, whereas the sodium ion ionophore ETH157 did not affect ATP formation in this system. Thus, we conclude that Ech hydrogenase acts as primary proton pump in a ferredoxin-dependent electron transport system.