The ASCTs (Alanine, Serine, Cysteine Transporters) belong to the Solute Carrier Family 1 (SLC1), which also includes the human glutamate transporters (Excitatory Amino Acid Transporters, EAATs) and the prokaryotic aspartate transporter GltPh. Despite the high degree of amino acid sequence identity between family members, ASCTs function quite differently from the EAATs and GltPh. We have used site-directed mutagenesis to investigate differences in substrate selectivity and ion coupling to better understand the structural basis for the different transport mechanisms of the SLC1 family. We have identified four key residues involved in determining differences in substrate selectivity between ASCT1, ASCT2, the EAATs and GltPh. Acidic amino acid transport by the EAATs is coupled to the co-transport of three Na+ ions and one proton, and the counter-transport of one K+ ion. In contrast, neutral amino acid exchange by the ASCTs does not require protons or the counter-transport of K+ ions and the number of Na+ ions required is not well established. We have investigated the number and location of Na+ ions required by ASCT1 by mutating residues in ASCT1 that correspond to residues in the EAATs and GltPh that are involved in Na+ binding. Mutations to all three proposed Na+ sites influence the binding of substrate and/or Na+, or the rate of substrate exchange. Thus, all three Na+ sites are conserved between different members of the family, but it also appears that not all three are essential for the substrate exchange process of ASCT1.