The ability of the two highly homologous Na⁺/Cl^--dependent neutral amino acid transporters - KAAT1 and CAATCH1, cloned from the midgut epithelium of the larva Manduca sexta- to transport different amino acids depends on the cotransported ion, on pH and on the membrane voltage. Different organic substrates give rise to transport-associated currents with their own characteristics, which are notably distinct between the two proteins. Differences in amplitude, kinetics and voltage-dependence of the transport-associated currents have been observed, as well as different substrate selectivity patterns measured by radioactive amino acid uptake assays. These diversities represent useful tools to investigate the structural determinants involved in the substrate selectivity. To identify these regions, four chimeric proteins between the two transporters were built. These proteins, heterologously expressed in Xenopus laevis oocytes, were analysed by two-electrode voltage clamp and uptake measurements. Initially, the first three domains were exchanged, obtaining the chimeras C3K9 and K3C9 (where the numbers indicate the transmembrane domains and the capital letters stay for the original proteins), which showed electrophysiological and [³H]amino acid uptake characteristics resembling those of KAAT1 and CAATCH1 respectively. The subsequent substitution of the last four domains in C3K9 and K3C9 gave the proteins C3K5C4 and K3C5K4 that showed the same behaviour of KAAT1 and CAATCH1 in electrophysiological and transport determinations. These results suggest that in KAAT1 and CAATCH1 only the central transmembrane domains (from 4 to 8) of the protein are responsible of the substrate selectivity.