A bumetanide-sensitive Na-K-Cl cotransporter (rBSC1) was recently cloned from a rat renal outer medulla (OM) cDNA library and shown to be expressed predominantly in the kidney. The purpose of the present study was to examine the nephron distribution of cotransporter transcripts and protein in rat kidney. In situ hybridization showed an intense signal only in the outer medulla and extending along cortical medullary rays consistent with expression of rBSC1 transcripts in medullary (MTAL) and cortical (CTAL) thick ascending limbs. Polyclonal antibodies raised in rabbits against a unique 67 amino acid segment from the carboxyl terminus of rBSC1 identified a broad major band of 130 to 160 (midpoint of 150) kDa and at least two minor bands of 50 to 70 kD on Western blotting of homogenates from cortex (C) and outer medulla (OM), but not inner medulla (IM), of rat kidney. Thus the Na-K-Cl cotransporter protein detected by the polyclonal rBSC1 antibody in rat kidney was similar in size to the major approximately 150 kD bumetanide binding protein detected by others in mouse and dog kidneys. Immunofluorescence studies using the anti-rBSC1 polyclonal antibody on rat kidney sections showed an intense signal limited to apical surfaces of MTAL and CTAL segments. Colocalization with anti-Tamm-Horsfall antibody which is present in all TABA cells except macula densa cells confirmed the absence of anti-rBSC1 fluorescence in the macula densa cells. These results are consistent with rBSC1 encoding the, or the major isoform of the, apical Na-K-Cl cotransporter in the thick ascending limb. The Na-K-Cl cotransporter functionally detected in macula densa cells may be encoded by a different BSC isoform.