We investigate the intrarenal expression of two recently cloned chloride channels, rClC-K1 and rClC-K2, by reverse transcriptase-polymerase chain reaction on single microdissected tubules from the rat kidney and by immunohistochemistry using a polyclonal antibody that recognizes both highly homologous channels. Both rClC-K1 and rClC-K2 mRNAs were detected in outer medullary late proximal tubules (S3), papillary ascending thin limbs (ATL), and outer medullary (MTAL) and cortical (CTAL) thick ascending limbs, distal tubules (DCT), and cortical, outer medullary, and inner medullary collecting ducts. Indirect immunofluorescence studies demonstrated that the rClC-K proteins were restricted to the basolateral membranes from ATL, DCT, and collecting ducts cells, whereas CTAL and MTAL exhibited a more diffuse basal staining. When rats were dehydrated, a condition which increased the expression of rClC-K1 in cortex and medulla, a weak cytoplasmic staining was found in late proximal tubule cells. Thus these results demonstrate that rat kidney ClC-K channels are predominantly located in the basolateral membranes from cells of the late segments of the renal tubule where most of chloride reabsorption takes place.