A transport system for branched-chain amino acids (designated as LIV-II system) in Pseudomonas aeruginosa requires Na+ for its operation. Coupling cation for this system was identified by measuring cation movement during substrate entry using cation-selective electrodes. Uptakes of Na+ and Li+ were induced by the imposition of an inwardly-directed concentration gradient of leucine, isoleucine, or valine. No uptake of H+ was found, however, under the same conditions. In addition, effects of Na+ and Li+ on the kinetic property of the system were examined. At chloride salt concentration of 2.5 mM, values of apparent Km and Vmax for leucine uptake were larger in the presence of Na+ than Li+. These results indicate that the LIV-II transport system is a Na+(Li+)/substrate cotransport system, although effects of Na+ and Li+ on kinetics of the system are different.