An aspartate:alanine antiporter (AspT) from the lactic acid bacterium Tetragenococcus halophilus catalyzes the electrogenic aspartate1-:alanine0 exchange reaction. Our previous kinetic analyses of transport reactions mediated by AspT in reconstituted liposomes suggested that, although the substrate transport reactions are physiologically coupled, the putative binding sites of l-aspartate (-Asp) and l-alanine (-Ala) are independently located on AspT. By using the fluorescent probe Oregon Green maleimide (OGM), which reacts specifically with cysteine, we also found that the presence of l-Asp changes the conformation of AspT. In this study, we conducted an OGM labeling assay in the presence of l-Ala. The labeling efficiency of single cysteine mutants (G62C and P79C) in transmembrane helix 3 of the AspT showed novel patterns depending on the presence of l-Ala or analogs. A concentration-dependent shift of AspT from the conformation in the presence of one substrate to that specific to the substrate added subsequently (l-Ala or l-Asp) was observed. Moreover, size-exclusion-chromatography-based thermostability assays indicated that the thermal stability of AspT in the presence of l-Ala differed from that in the presence of l-Asp. From these results, we concluded that l-Ala binding yields a conformation different from the apo or l-Asp binding conformations.