The human glutamine/neutral amino acid transporter ASCT2 (hASCT2) was over-expressed in P. pastoris and purified by Ni(2+)-chelating and gel filtration chromatography. The purified protein was reconstituted in liposomes by detergent removal with a batch-wise procedure. Time dependent [(3)H]glutamine/glutamine antiport was measured in proteoliposomes which was active only in the presence of external Na(+). Internal Na(+) slightly stimulated the antiport. Optimal activity was found at pH 7.0. A substantial inhibition of the transport was observed by Cys, Thr, Ser, Ala, Asn and Met (≥ 70%) and by mercurials and methanethiosulfonates (≥ 80%). Heterologous antiport of [(3)H]glutamine with other neutral amino acids was also studied. The transporter showed asymmetric specificity for amino acids: Ala, Cys, Val, Met were only inwardly transported, while Gln, Ser, Asn, and Thr were transported bi-directionally. From kinetic analysis of [(3)H]glutamine/glutamine antiport Km values of 0.097 and 1.8 mM were measured on the external and internal sides of proteoliposomes, respectively. The Km for Na(+) on the external side was 32 mM. The homology structural model of the hASCT2 protein was built using as template the GltPh of P. horikoshii. Cys395 was the only Cys residue externally exposed, thus being the potential target of SH reagents inhibition and, hence, potentially involved in the transport mechanism.