The Na-K-Cl cotransporter (NKCC) is present in most animal cells where it functions in cell volume homeostasis and epithelial salt transport. We developed six monoclonal antibodies (designated T4, T8, T9, T10, T12, and T14) against a fusion protein fragment encompassing the carboxy-terminal 310 amino acids of the human colonic NKCC. These T antibodies selectively recognized putative NKCC proteins in a diverse variety of animal tissues. Western blot analysis of membranes isolated from 23 types of cells identified single bands of immunoreactive protein ranging in mass from 146 to 205 kDa. The amount of immunoreactive protein detected in these cells correlated with loop diuretic binding site density. Proteins identified previously as Na-K-Cl cotransporters by loop diuretic photoaffinity labeling were mutually recognized by multiple T antibodies. Most of the T antibodies effectively immunoprecipitated the denatured form of the NKCC protein. Immunocytochemical studies on the rabbit parotid gland demonstrated that NKCC is restricted to the basolateral margin of the acinar cells and absent from the ducts, in accord with the central role of Na-K-Cl cotransport in chloride secretion. In the rabbit kidney, NKCC was localized to the apical membrane of thick ascending limb cells, consistent with its role in chloride reabsorption.