Hepatocyte nuclear factor 1 is a family of transcription factors that contain an extra-long homeodomain and are involved in tissue-specific gene expression in liver and intestine. One family member (HNF-1α) may also be important for epithelial cell differentiation. During nephrogenesis, expression of HNF-1α coincides with induction of the kidney-specific Na-K-Cl cotransporter (Nkcc2). We cloned the proximal 5′ flanking region of the murine Nkcc2 gene and identified an element located 212 bp 5′ to the transcription initiation site that was identical at 13/15 nt with a consensus HNF-1 binding site. To determine whether HNF-1α could bind to this sequence in vitro, electrophoretic mobility-shift assays were performed using a radiolabeled probe corresponding to nt -271 to -3 of the proximal Nkcc2 5′ flanking region. Murine HNF-1α, synthesized using a coupled transcription-translation reaction, was able to retard the mobility of the-271/-3 fragment in a dose-dependent manner. Binding was reduced in the presence of excess unlabeled -271/-3 fragment but was unaffected by an excess of poly(dIdC)(dIdC). Binding was also eliminated by a 1000-fold molar excess of an oligonucleotide containing either the wild-type sequence of the putative HNF-1 site at -212 or a known HNF-1 site from the rat β-fibrinogen promoter (β-28). In contrast, a 1000-fold excess of an oligonucleotide containing two point mutations of the putative HNF-1 site at -212 was unable to compete for binding. To test whether binding of HNF-1α to the site at-212 could stimulate promoter activity, transient co-transfections were performed. The -271/-3 fragment was cloned into a plasmid containing luciferase driven by a minimal SV40 promoter then transfected into NIH 3T3 cells which do not express endogenous HNF-1α. Co-transfection with an HNF-1α expression plasmid resulted in a 2.5-fold stimulation of luciferase activity. These results indicate that the proximal 5′ flanking region of the Nkcc2 gene contains a functional HNF-1α binding site. HNF-1α may contribute to kidney-specific expression ofNkcc2.