A low K-resistant mutant Madin-Darby canine kidney (MDCK) cell line, LK-C1, has been shown previously to lack functional Na-K-Cl cotransporter (NKCC) activity, indicating that it may be a useful NKCC "knockout" cell line for structure-function studies. Using immunological probes, we first characterized the defect in the endogenous NKCC protein of the LK-C1 cells and then fully restored NKCC activity in these cells by stably expressing the human secretory NKCC1 protein (hNKCC1). The endogenous NKCC protein of the LK-C1 cells was expressed at significantly lower levels than in wild-type MDCK cells and was not properly glycosylated. This latter finding indicated that the lack of functional NKCC activity in the LK-C1 cells may be due to the inability to process the protein to the plasma membrane. In contrast, exogenously expressed hNKCC1 protein was properly processed and fully functional at the plasma membrane. Significantly, the exogenous hNKCC1 protein was regulated in a manner similar to the protein in native secretory cells as it was robustly activated by cell shrinkage, calyculin A, and low-Cl incubation. Furthermore, when the LK-C1 cells formed an epithelium on permeable supports, the exogenous hNKCC1 protein was properly polarized and functional at the basolateral membrane. The low levels of endogenous NKCC protein expression, the absence of any endogenous NKCC transport activity, and the ability to form a polarized epithelium indicate that the LK-C1 cells offer an excellent expression system with which to study the molecular physiology of the cation Cl cotransporters.