A gene encoding a Li(+) extrusion system was cloned from the chromosomal DNA of Pseudomonas aeruginosa and expressed in Escherichia coli cells. The gene enabled growth of E. coli KNabc cells, which were unable to grow in the presence of 10 mM LiCl or 0.1 M NaCl because of the lack of major Na(+) (Li(+))/H(+) antiporters. We detected Li(+)/H(+) and Na(+)/H(+) antiport activities in membrane vesicles prepared from E. coli KNabc cells that harbored a plasmid carrying the cloned gene. Activity of this antiporter was pH-dependent with an optimal pH activity between pH 7.5 and 8.5. These properties indicate that this antiporter is different from NhaP, an Na(+)/H(+) antiporter from P. aeruginosa that we reported previously, and that is rather specific to Na(+) but it cannot extrude Li(+) effectively. The gene was sequenced and an open reading frame (ORF) was identified. The amino acid sequence deduced from the ORF showed homology (about 60% identity and 90% similarity) with that of the NhaB Na(+)/H(+) antiporters of E. coli and Vibrio parahaemolyticus. Thus, we designated the antiporter as NhaB of P. aeruginosa. E. coli KNabc carrying the nhaB gene from P. aeruginosa was able to grow in the presence of 10 to 50 mM LiCl, although KNabc carrying nhaP was unable to grow in these conditions. The antiport activity of NhaB from P. aeruginosa was produced in E. coli and showed apparent Km values for Li(+) and Na(+) of 2.0 mM and 1.3 mM, respectively. The antiport activity was inhibited by amiloride with a Ki value for Li(+) and Na(+) of 0.03 mM and 0.04 mM, respectively.