CLC-K1 and CLC-K2, two kidney-specific CLC chloride channels, are transcriptionally regulated on a tissue-specific basis. We have shown that a GA element near their transcriptional start sites is important for basal and cell-specific activities of the CLC-K1 and CLC-K2 gene promoters. To identify the GA-binding proteins, a kidney cDNA library was screened by a yeast one-hybrid system. A novel member of the Cys2-His2 zinc finger gene designated as KKLF (kidney-enriched Krüppel-like factor) and the myc-associated zinc finger protein (MAZ) were cloned. KKLF was found to be abundantly expressed in the liver, kidney, heart, and skeletal muscle. In the kidney, KKLF protein was localized in interstitial cells, mesangial cells, and nephron segments where CLC-K1 and CLC-K2 were not expressed. Gel mobility shift assay revealed that recombinant KKLF and MAZ proteins exhibited sequence-specific binding to the CLC-K1 GA element and that the consensus sequence for the KKLF binding site was GGGGNGGNG. In transient transfection, MAZ had a strong activating effect on the CLC-K1-luciferase reporter gene transcription. On the other hand, KKLF coexpression with MAZ appeared to block the activating effect of MAZ. These results suggest that a novel set of zinc finger proteins may help regulate the strict tissue and nephron segment-specific expression of CLC-K1 and CLC-K2 channel genes through their GA cis element.