We have recently isolated cDNAs encoding a Na(+)-H+ exchanger isoform, referred to as NHE-1, from rabbit kidney and LLC-PK1 cells. To identify the NHE-1 protein and to establish its cellular and subcellular localization in the rabbit kidney, we prepared antibodies to a NHE-1 fusion protein. cDNA encoding the COOH-terminal 41 amino acids of NHE-1 was subcloned into a maltose-binding protein vector and the purified fusion protein (FP347A) used to immunize guinea pigs. To identify the NHE-1 protein, we performed Western blot analysis against membrane fractions prepared from rabbit renal cortex. Anti-FP347A antibody specifically reacted with a polypeptide with an apparent molecular mass of 100–110 kDa that was enriched in basolateral membrane fractions. When indirect immunofluorescence was performed on semithin (0.5 micron) cryosections of paraformaldehyde-lysine-periodate-fixed rabbit kidney, anti-FP347A specifically stained the basolateral plasma membrane of cells of the proximal tubule, thick ascending limb, and distal convoluted tubule. Anti-FP347A similarly stained connecting tubule cells and principal cells. No staining was detected on the apical membrane of any cells of the rabbit nephron. We conclude that NHE-1 is a 100- to 110-kDa protein expressed on the basolateral membrane of multiple nephron segments.