The mammalian kidney bumetanide-sensitive Na+-K+-2Cl−and thiazide-sensitive Na+-Cl−cotransporters are the major pathways for salt reabsorption in the thick ascending limb of Henle's loop and distal convoluted tubule, respectively. These cotransporters serve as receptors for the loop- and thiazide-type diuretics, and inactivating mutations of corresponding genes are associated with development of Bartter's syndrome type I and Gitleman's disease, respectively. Structural requirements for ion translocation and diuretic binding specificity are unknown. As an initial approach for analyzing structural determinants conferring ion or diuretic preferences in these cotransporters, we exploited functional differences and structural similarities between Na+-K+-2Cl−and Na+-Cl−cotransporters to design and study chimeric proteins in which the NH2-terminal and/or COOH-terminal domains were switched between each other. Thus six chimeric proteins were produced. Using the heterologous expression system of Xenopus laevis oocytes, we observed that four chimeras exhibited functional activity. Our results revealed that, in the Na+-K+-2Cl−cotransporter, ion translocation and diuretic binding specificity are determined by the central hydrophobic domain. Thus NH2-terminal and COOH-terminal domains do not play a role in defining these properties. A similar conclusion can be suggested for the Na+-Cl−cotransporter.