The Alanine, Serine, Cysteine Transporters (ASCTs) belong to the Solute Carrier Family 1A (SLC1A), along with the human glutamate transporters (Excitatory Amino Acid Transporters, EAATs). ASCTs exchange neutral amino acids in an electroneutral fashion, while the EAATs transport acidic amino acids, generating a net flux of substrate and charge. Both ASCT- and EAAT-mediated transport are Na⁺-dependent, however Na⁺ binding and coupling in ASCTs has not been well established. The co-ordinating residues of the three established Na⁺ sites in the EAATs are conserved in ASCTs. We therefore initiated a study of ASCT1 to investigate Na⁺ binding and coupling to neutral amino acid exchange. Residues involved in Na⁺ binding in the EAATs were targeted for mutagenesis in ASCT1. Mutations of D467 probed the Na1 site, G422 the Na2 site, and D380 the Na3 site. ASCT1 transporters were expressed in Xenopus laevis oocytes and analysed using electrophysiology and radiolabelled uptake experiments. We probed the existence of a Na2 site by generating G422S in ASCT1. The G422S mutant transporter displayed similar apparent L-serine affinity and uptake levels compared to wild type, however a reduced apparent Na⁺ affinity. Neutralising the aspartate residues in Na1 or Na3 (D467N, D380N respectively) generates transporters that resemble wild type ASCT1in substrate binding (K_0.5=100±16µM), Na⁺ binding (K_0.5=46±3mM) and levels of ³H-L-serine uptake (1420±70fmol/oocyte/min). Less conservative mutations in Na1 (D467A) maintain Na⁺ binding, however impair substrate binding (K_0.5=800±200µM). D380A, in the Na3 site, maintains substrate binding, however impairs Na⁺ binding (K_0.5=240±70mM). Interestingly, both D467A and D380A mutant transporters do not exchange ³H-L-serine at levels above background. This suggests that Na1, Na2 and Na3 are conserved between the EAATs and ASCT1. At least one of Na1 or Na3 is required for exchange; however Na1 and Na3 are not required for activation of the anion conductance, which is in contrast to the EAATs.