ATPase activity of the membrane-bound Na(+)-ATPase of an alkaliphilic bacterium, Exiguobacterium aurantiacum, was measured in various concentrations of NaCl. Hill plot analysis showed a Hill number of 1.7 with 5.2 mM as the K(0.5) value for Na(+). When the site-directed mutagenesis of seven charged amino acid residues in the cytoplasmic loop (L6/7) between putative transmembrane segments 6 and 7 of the enzyme was conducted, all the mutated enzymes exhibited Hill numbers close to that of the wild-type enzyme (WT). When reconstituted with lecithin, all the mutants exhibited Na(+)-transport activity. While alanine substitution for several residues gave some significant effects on the enzyme function, the most remarkable effect was observed in the substitution for Glu-733. The K(0.5) value of E733A for Na(+) was 83.2 mM. The mutant exhibited only 8.5% of the ATPase activity and 54.0% of the energy-coupling efficiency for Na(+) transport as compared with those of WT, respectively. Drastic decreases of apparent affinity for Na(+) and energy efficiency of ion transport were also observed in E733K and E733T, respectively.