The cells of Pseudomonas aeruginosa showed high activity for leucine transport in the absence of Na+, giving a Km value of 0.34 microM. In the presence of Na+, however, two Km values, 0.37 microM (LIV-I system) and 7.6 microM (LIV-II system), were obtained. The former system seemed to serve not only for the entry of leucine, isoleucine, and valine, but also for that of alanine and threonine, although less effectively. However, the LIV-II system served for the entry of branched-chain amino acids only. The LIV-II system alone was operative in membrane vesicles, for the transport of branched-chain amino acids in membrane vesicles required Na+ and gave single Km values for the respective amino acids. When cells were osmotically shocked, the activity of the LIV-I system decreased, whereas the LIV-II system remained unaffected. The shock fluid from P. aeruginosa cells showed leucine-binding activity with a dissociation constant of 0.25 microM. The specificity of the activity was very similar to that of the LIV-I system. These results suggest that a leucine-binding protein(s) in the periplasmic space may be required for the transport process via the LIV-I system of P. aeruginosa.