Most neutral L-amino acid acids are transported actively across the luminal brush border membrane of small intestine and kidney proximal tubule epithelial cells by a Na⁺ co-transport system named B^O that has been recently molecularly identified (B^OAT1, SLC6A19). We show here that the opossum kidney-derived cell line OK also displays a Na⁺-dependent B^O-type neutral L-amino acid transport, though with a slightly differing substrate selectivity. We tested the hypothesis that one of the two B^OAT1-related transporters, SLC6A18 (ortholog of orphan transporter XT2) or SLC6A20 (orthologue of the recently identified mammalian imino acid transporter SIT1), mediates this transport. Anti-sense RNA to OK SIT1 (oSIT1) but not to OK XT2 (oXT2) inhibited Na⁺-dependent neutral amino acid transport induced by OK mRNA injected in Xenopus laevis oocytes. Furthermore, inhibition of oSIT1 gene expression in OK cells by transfection of siRNA and expression of shRNA selectively reduced the Na⁺-dependent uptake of neutral L-amino acids. Finally, expression of OK cell oSIT1 cRNA in Xenopus oocytes induced besides the transport of the L-imino acid L-Pro also that of neutral L-amino acids. Taken together the data indicates that in opossum kidney OK cells SIT1 (SLC6A20) is not only an apical imino acid transporter but also plays a major role as Na⁺-dependent neutral L-amino acid transporter. A similar double role could be envisaged for SIT1 in mammalian kidney proximal tubule and small intestine.