The aspartate: alanine antiporter (AspT) of the lactic acid bacterium Tetragenococcus halophilus is a member of the aspartate: alanine exchanger (AAEx) transporter family. T. halophilus AspT catalyzes the electrogenic exchange of L-aspartate 1- with L-alanine 0. Although physiological functions of AspT were well studied, L-aspartate 1-:L-alanine 0 antiport mechanisms are still unsolved. Here we report that the binding sites of L-aspartate and L-alanine are independently present in AspT by means of the kinetic studies. We purified His 6-tagged T. halophilus AspT and characterized its kinetic properties when reconstituted in liposomes (K m = 0.35 ± 0.03 mM for L-aspartate, K m ± 0.098 ± 0 mM for D-aspartate, K m±26±2 mM for L-alanine, K m=3.3 ± 0.2 mM for D-alanine). Competitive inhibition by various amino acids of L-aspartate or L-alanine in self-exchange reactions revealed that L-cysteine selectively inhibited L-aspartate self-exchange but only weakly inhibited L-alanine self-exchange. Additionally, L-serine selectively inhibited L-alanine self-exchange but barely inhibited L-aspartate self-exchange. The aspartate analogs L-cysteine sulfinic acid, L-cysteic acid, and D-cysteic acid competitively and strongly inhibited L-aspartate self-exchange compared with L-alanine self-exchange. Taken together, these kinetic data suggest that the putative binding sites of L-aspartate and L-alanine are independently located in the substrate translocation pathway of AspT.