d,lthreo-β-Benzyloxyaspartate (d,l-TBOA), an analog of threo-β-hydroxyaspartate (THA) possessing a bulky substituent, is a potent non-transportable blocker for the excitatory amino acid transporters, EAAT1, 2 and 3, while lthreo-β-methoxyaspartate (l-TMOA) is a blocker for EAAT2, but a substrate for EAAT1 and EAAT3. To characterize the actions of these THA analogs and the function of EAAT4 and EAAT5, we performed electrophysiological analyses in EAAT4 or EAAT5 expressed on Xenopus oocytes. In EAAT4-expressing oocytes, d,l-TBOA acted as a non-transportable blocker, while l-TMOA like d,l-THA was a competitive substrate. In contrast, d,l-THA, d,l-TBOA and l-TMOA all strongly attenuated the glutamate-induced currents generated by EAAT5. Among them, l-TMOA showed the most potent inhibitory action. Moreover, d,l-THA, d,l-TBOA and l-TMOA themselves elicited outward currents at negative potentials and remained inward at positive potentials suggesting that d,l-TBOA and l-TMOA, as well as d,l-THA, not only act as non-transportable blockers, but also block the EAAT5 leak currents. These results indicate that EAATs 4 and 5 show different sensitivities to THA analogs although they share properties of a glutamate-gated chloride channel.