The present study was undertaken to investigate the mRNA localization of the two major kidney-specific Na-K-Cl transport proteins, the bumetanide-sensitive cotransporter (NKCC2 in rabbit and BSC1 in rat) and the thiazide-sensitive cotransporter (TSC). NKCC2 from rabbit and mouse has been shown to exist in three isoforms (designated A, B, and F) that differ only in a 96-bp region. The divergent region of each of the three NKCC2 isoforms was cloned from rat kidney by a polymerase chain reaction (PCR)-based strategy, and isoform-specific primers were chosen. RNA and cDNA were prepared from renal cortex and medulla and from microdissected nephron segments. Using reverse transcription (RT)-PCR, the B isoform was detected only in cortex and the F isoform only in medulla, whereas the A from was found in both. In dissected nephron segments, the B form was found exclusively in cortical thick ascending limb (CTAL) and macula densa-containing segment (MDCS), the F form only in medullary thick ascending limb (MTAL) and outer medullary collecting duct, and the A form in CTAL, MDCS, and MTAL. An additional isoform including both A and F exons was identified by direct sequencing of a 592-bp product from medulla. The AF product was found only in the medulla and was localized exclusively in MTAL. TSC mRNA was detected exclusively in the distal convoluted tubule. Differential nephron localization of NKCC2 isoforms suggests that Na-K-Cl cotransporters may differ in their transport characteristics to explain regulation of salt transport along the nephron.