The liv gene cluster responsible for encoding the high-affinity branched-chain amino acid transport proteins in Salmonella typhimurium was mapped in the 7.6-kilobase HindIII-SacI segment of plasmid pMN12 by utilizing the gene dosage effect. By subcloning and biochemical analysis, the livB and livC structural genes encoding the leucine-, isoleucine-, valine-, threonine-binding protein (LIVT-BP.) and the leucine-specific binding protein (L-BP.), respectively, were localized within the 3, 617-base HindIII-BstEII segment. Upon determining the nucleotide sequence of the 3, 617 bases, we found that the coding sequence of the livB gene (1, 095 base pairs) starts at the position 355 and specifies the precursor LIVT-BP of 365 amino acid residues, and the livC gene (1, 107 base pairs) starts at the position 2, 452 and encodes the precursor L-BP of 369 amino acid residues. The two genes, separated by a 1-kilobase intergenic region, each possess potential promoters and rhoindependent transcriptional terminators. The mature LIVT-BP and L-BP are produced by removing the putative 21 and 23 signal peptides from the respective precursors. In comparison with the analogous two binding proteins from Escherichia coli K-12, strong homologies are observed.