To establish a system to analyze ATP synthesis by the archaeal A1Ao ATP synthase and to address the nature of the coupling ion, the operon encoding the A1Ao ATP synthase from the mesophile Methanosarcina mazei Gö1 was cloned in an expression vector and it was expressed in the F1Fo ATP synthase-negative mutant Escherichia coli DK8. Western blot analyses revealed that each of the subunits was produced, and the subunits assembled to a functional, membrane-embedded ATP synthase/ATPase. ATP hydrolysis was inhibited by dicyclohexylcarbodiimide but also by tributyltin, which turned out to be the most efficient inhibitor of the Ao domain of A1Ao ATP synthase known to date. ATP hydrolysis was not dependent on the Na+ concentration of the medium, and inhibition of the enzyme by dicyclohexylcarbodiimide could not be relieved by Na+. The enzyme present in the cytoplasmic membrane of E. coli catalyzed ATP synthesis driven by an artificial ΔpH but not by ΔpNa or ΔμNa+.