V-ATPases make up a family of proton pumps distributed widely from bacteria to higher organisms. We found a variant of this family, a Na(+)-translocating ATPase, in a Gram-positive bacterium, Enterococcus hirae. The Na(+)-ATPase was encoded by nine ntp genes from F to D in an ntp operon (ntpFIKECGABDHJ): 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 the transcriptional level by intracellular Na(+) as the signal. Structural aspects and catalytic properties of purified Na(+)-ATPase closely resembled those of other V-type H(+)-ATPases. Interestingly, the E. hirae enzyme showed a very high affinity for Na(+) at catalytic reaction. This property enabled the measurement of ion binding to this ATPase for the first time in the study of V- and F-ATPases. Properties of Na(+) binding to V-ATPase were consistent with the model that V-ATPase proteolipids form a rotor ring consisting of hexamers, each having one cation binding site. We propose here a structure model of Na(+) binding sites of the enzyme.