Isolated membranes of the moderate halophilic bacterium Haloferax volcanii are able to hydrolyze ATP via an ATPase, which needs the presence of Mg 2+ or Mn 2+ , high concentra tions of NaCl, a pH value of 9, and high temperatures with an optimum at 60 °C. We have not found any phosphatase activity in the preparations. We developed a purification method for the isolated enzyme with an enrichment factor of 90. SDS-gel electrophoresis of the partially purified enzyme of Haloferax volcanii showed putative ATPase subunits of 63, 51, 37, and 12 kDa. N-ethylmaleimide (NEM) a specific inhibitor for V-ATPases, which alkylates cysteines, inhibited the enzyme slightly. Binding of tritiated NEM to the isolated ATPase fractions resulted in labelling of the 63 and 51 kDa peptides. Using PCR with degenerate oligonucleotides, we could clone and sequence a gene cluster encoding the Aj part of the halophilic ATPase. The described genes are organized in an operon in the order D. C, E, B, A , named alphabetically according to their decreasing size. The deduced products of 64.5, 52, 38.7, 22, and 11.6 kDa confirm the results of the partial purification of the ATPase. Biochemical characterization of the Haloferax volcanii ATPase gave the following results: In presence of Mn 2+ higher rates of ATP hydrolysis could be observed than in presence of Mg 2+ , but free manganese ions inhibited the enzyme activity of the ATPase. Calculation of the true concentrations of the complex between ATP and the respective divalent metal ion led to determination of Michaelis-Menten constants for ATP in the hydrolysis direction of 1 mм in presence of MgCl 2 and 0.24 mм in presence of MnCl 2 . Sodium chloride concentrations in the molar range induce changes in K M by a factor of about 10. The enzyme is specific for ATP; other nucleotides including GTP and ADP are competitive inhibitors of ATP hy drolysis.