The histidine ATP-binding cassette (ABC) transporter of Salmonella enterica serovar Typhimurium is among the best-studied type I ABC import systems. The transporter consists of two transmembrane subunits, HisQ and HisM, and a homodimer of the nucleotide-binding subunit, HisP. Substrates are delivered by two periplasmic solute binding proteins, HisJ and LAO, with preferences for histidine and for lysine, arginine, and ornithine, respectively. A homology model was built by using the arginine-bound crystal structure of the closely related Art(QN)2 transporter of Thermoanaerobacter tengcongensis as the template. In the homodimeric Art(QN)2, one substrate molecule is bound to each of the ArtQ subunits, whereas the structural model and sequence alignments predict only one substrate molecule in contact with HisM. To address the question whether one or two binding sites exist in heterodimeric HisQM, we have studied the functional consequences of mutations by monitoring (i) the complementation of growth on d-histidine of auxotrophic tester strains, (ii) the growth of tester strains on arginine as a nitrogen source, and (iii) ATPase activity of purified variants in a lipid environment. Our results demonstrate that two negatively charged residues, namely, HisM-E166 and HisQ-D61, are indispensable for function. Furthermore, the complete reconstruction of an ArtQ-like binding site in HisQ resulted in an inactive transporter. Likewise, switching the positions of both negatively charged residues between HisQ and HisM caused transport-deficient phenotypes. Thus, we propose that one substrate molecule is primarily liganded by residues of HisM while HisQ-D61 forms a crucial salt bridge with the α-amino group of the substrate.IMPORTANCE Canonical ATP-binding cassette (ABC) importers are major players in the translocation of numerous nutrients, vitamins, and growth factors to the cytoplasm of prokaryotes. Moreover, some ABC importers have been identified as virulence factors in bacterial pathogenesis. Thus, a full understanding of their mode of action is considered a prerequisite, among others, for the development of novel antibacterial drugs. However, mainly owing to the lack of structural information, the knowledge of the chemical nature and number of substrate binding sites formed by the transmembrane subunits of ABC importers is scarce. Here, we provide evidence from mutational analyses that, in contrast to homologous homodimeric systems, the heterodimeric histidine transporter of Salmonella enterica serovar Typhimurium is liganding only one substrate molecule between its transmembrane subunits, HisM and HisQ.