In Escherichia coli, the active transport of branched-chain amino acids was performed by three different kinetically system: Leucine-isoleucine-valine (LIV)- I, II and Leucine-specific (LS) system. The transport capacity of LS system depends on a periplasmic protein, leucine-specific binding protein (LS-BP). In previous studies, the substrate specificity of LS-BP was revealed in vivo, but the mechanism of substrate recognition remains unclear. In this study, we purified LS-BP and measured the affinity for leucine and its derivatives by BIACORE. Since the affinity of LS-BP for leucine and its derivatives exceeded the maximum range of BIACORE, the Km value could not be determined. Then we developed an in vitro assay to investigate the substrate recognition of LS-BP by using radiolabeled leucine. In this assay, the derivatives modified with NH₂, COO, or Cγ did not show an obvious binding to LS-BP, while no significant differences were observed between leucine and its derivatives modified with OH, Cα or Cβ. Those results suggest that LS-BP recognizes NH₂, COO, and Cγ of leucine.