Na-K-Cl cotransport plays an important role in the kidney in NaCl reabsorption in the thick ascending limb of Henle and a less well defined role in the inner medullary collecting duct (IMCD). Two Na-K-Cl cotransporters encoded by different genes have been identified in the mammalian kidney: BSC1/NKCC2 which localizes to the apical thick ascending limb of Henle and BSC2/NKCC1 which was isolated from a mouse IMCD cell line (mIMCD-3) but its localization has not been determined. In this study we generated a polyclonal antibody (anti-mBSC2) against the mouse BSC2/NKCC1 protein in order to characterize and localize this protein in mouse kidney. Western blot analysis with affinity-purified anti-mBSC2 showed a protein doublet of 140 and 150 kD which was most abundant in the renal papilla but also seen in cortex and outer medulla. The 140-150-kD bands were not seen with preimmune serum or with anti-mBSC2 preabsorbed with specific antigen. Immunolocalization confirmed expression of mBSC2 protein on the basolateral surface of terminal IMCD segments and demonstrated expression in the papillary surface epithelium. Immunofluorescence also revealed the unexpected presence of the BSC2 protein at the juxtaglomerular afferent arteriole, in a juxtaglomerular structure probably representing the extraglomerular mesangium, and throughout the glomerular mesangium.