A Na+-translocating ATPase was discovered in a gram-positive bacterium Enterococcus hirae. Our biochemical and molecular biological studies revealed that this Na+-ATPase belongs to the vacuolar-type enzyme. Purified Na+-ATPase consisted of nine subunits: NtpA, B, C, D, E, F, G, I, and K; reconstituted proteoliposomes showed ATP-driven electrogenic Na+ translocation. All these subunits were encoded by the ntp operon: ntpFIKECGABDHJ. The deduced amino acid sequences of the major subunits, A, B, and K (16 kDa proteolipid), were highly similar to those of A, B, and proteolipid subunits of vacuolar ATPases, although the similarities of other subunits were moderate. The ntpJ gene encoded a K+ transporter independent of the Na+-ATPase. Expression of this operon, encoding two transport systems for Na+ and K+ ions, was regulated at transcriptional level by intracellular Na+ as the signal. Two related cation pumps, vacuolar Na+-ATPase and F0F1, H+-ATPase, coexist in this bacterium.