The Gram-positive bacteria Enterococcus hirae expel sodium by two systems: a Na+/H(+)-antiporter and a vacuolar-type Na(+)-ATPase. We isolated a mutant, NalkA, defective in the Na(+)-ATPase. NalkA grew normally at neutral pH but was unable to grow in the presence of > 100 mM sodium at pH 9.5. By functional complementation at high pH, we cloned pES1, a plasmid from an E. hirae gene bank containing a 5.2-kilobase pair region of genomic DNA. The genomic DNA in pES1 contains five complete open reading frames, ntpM, -N, -O, -P, and -Q, encoding proteins of 75, 16, 23, 38, and 11 kDa. A sixth incomplete open reading frame, ntp 'L, precedes ntpM. The 3'-end of the cloned DNA overlaps with a previously published sequence encoding the ntpA and ntpB subunits of the E. hirae Na(+)-ATPase (Takase, K., Yamato, I., and Kakinuma, Y. 1993) J. Biol. Chem. 268, 11610-11616). The insert of pES1 therefore represents the upstream region of the ntp operon that encodes the E. hirae Na(+)-ATPase. Complementation analysis with various deletions derived from pES1 suggest that the original mutation is in the ntpM gene. Of the new genes described here, three exhibited significant sequence similarity to known proteins; ntpM shares 24% identical amino acid residues with the "116-kDa" subunits of eukaryotic vacuolar ATPases, ntpN exhibits 28% sequence identity with the 16-kDa proteolipid of human vacuolar ATPase, and ntpO has sequence homology to the 31-kDa subunit of the bovine kidney vacuolar ATPase. No known proteins with sequence similarity to ntp'L, -P, or -Q could be identified. Disruption of either ntpM, -N, or -O in wild-type cells by cassette mutagenesis resulted in mutants unable to effect ATP-driven sodium extrusion. NtpM, -N, and -O therefore represent three new gene products involved in sodium extrusion by the vacuolar-type Na(+)-ATPase of E. hirae, and three more gene products, NtpL, -P, and -Q, may also be constituents of this enzyme. The ntp operon thus contains at least eight genes.