Two kidney-specific chloride channels, ClC-K1 and ClC-K2, have been isolated from rat kidney. In the present study, we sought to isolate human homologue of rat ClC-K2 chloride channel that was present in the thick ascending limb of Henle's loop and collecting ducts. Human kidney cDNA library was screened with the whole rat ClC-K2 cDNA probe. Two highly homologous but not identical cDNAs were isolated and sequenced. Northern analysis showed that both clones were expressed only in kidney among various human tissues, demonstrating that kidney-specific ClC family members were also present in human kidney. Because both clones had almost the same nucleotide identity (approximately 80%) with rat ClC-K2, we could not determine by sequence alone which human clone corresponded to rat ClC-K2. Accordingly, we performed reverse transcription PCR using dissected human nephron segments and identified the site of expression of each clone in human nephron segments. One clone was only expressed in the thin limb of Henle's loop and the other was expressed in glomeruli, proximal tubules, and collecting ducts. We identified the latter clone as human ClC-K2 based on the localization of rat ClC-K1 and ClC-K2. Identification of human ClC-K2 clone will be of help in understanding the genetic involvement of chloride channel in disorders of chloride transport such as Bartter's syndrome.