The ntpJ gene, the tail end in the vacuolar type Na+-ATPase (ntp) operon of Enterococcus hirae, encodes a putative 49-kDa hydrophobic protein resembling K+ transporter protein in Saccharomyces cerevisiae (Takase, K., Kakinuma, S., Yamato, I., Konishi, K., Igarashi, K., and Kakinuma, Y. (1994) J. Biol. Chem. 269, 11037-11044). Northern blotting experiment revealed that the ntpJ gene was transcribed as a cistron in the ntp operon. We constructed an Enterococcus strain in which the ntpJ gene was disrupted by cassette mutagenesis with erythromycin resistance gene. The growth of this mutant was normal at low pH. However, the mutant did not grow at high pH in K+-limited medium (less than 1 mM), while the wild type strain grew well; the internal K+ concentration of this mutant was as low as 7% of that of the wild type strain, suggesting that the K+ accumulation at high pH was inactivated by disruption of the ntpJ gene. Potassium uptake activity via the KtrII system, which had been proposed as the proton potential-independent, Na+-ATPase-coupled system working at high pH (Kakinuma, Y., and Harold, F. M. (1985) J. Biol. Chem. 260, 2086-2091), was missing in this mutant strain. However, this mutant retained as high activities of Na+-ATPase and Na+ pumping as the wild type strain. From these results, we conclude that the NtpJ is a membraneous component of the KtrII K+ uptake system but not a functional subunit of vacuolar Na+-ATPase complex; the interplay between the KtrII system and the Na+-ATPase was discussed.