Further evidence on the role of binding proteins in branched-chain amino acid transport in Escherichia coli was obtained by selecting mutants with altered expression of the binding proteins. The mutator phage Mu was used to induce E. coli L-valine-resistant mutants defective in branched-chain amino acid transport. By making use of mild selective conditions and strain backgrounds with derepressed high-affinity, binding protein-mediated transport systems, we were able to isolate a new class of transport mutants defective in these systems. Mutant strains AE84084 (livK::Mucts) and AE840102 (livJ) were found to be defective in leucine-specific and LIV binding proteins, respectively, by transport assay, in vitro binding activity, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mutant strain AE4107 (livH::Mu), although lacking high-affinity, branched-chain amino acid transport, retained functional binding proteins and therefore evidently codes for an additional component of high-affinity transport. The livH, livJ, and livK mutations were mapped by transduction and shown to be closely linked to each other in the malT region (min 74) of the E. coli genetic map.