The thiazide-sensitive Na+-Cl- cotransporter (NCC) is the major pathway for salt reabsorption in the distal convoluted tubule, serves as a receptor for thiazide-type diuretics, and is involved in inherited diseases associated with abnormal blood pressure. Little is known regarding the structure-function relationship in this cotransporter. Previous studies from our group reveal that mammalian NCC exhibits higher affinity for ions and thiazides than teleost NCC and suggest a role for glycosylation upon thiazide affinity. Here we have constructed a series of chimeric and mutant cDNAs between rat and flounder NCC to define the role of glycosylation status, the amino-terminal domain, the carboxyl-terminal domain, the extracellular glycosylated loop, and the transmembrane segments upon affinity for Na+, Cl-, and metolazone. Xenopus laevis oocytes were used as the heterologous expression system. We observed that elimination of glycosylation sites in flounder NCC did not affect the affinity of the cotransporter for metolazone. Also, swapping the amino-terminal domain, the carboxyl-terminal domain, the glycosylation sites, or the entire extracellular glycosylation loop between rat and flounder NCC had no effect upon ions or metolazone affinity. In contrast, interchanging transmembrane regions between rat and flounder NCC revealed that affinity-modifying residues for chloride are located within the transmembrane 1-7 region and for thiazides are located within the transmembrane 8-12 region, whereas both segments seem to be implicated in defining sodium affinity. These observations strongly suggest that binding sites for chloride and thiazide in NCC are different.