Mammalian hepatocyte nuclear factor-3 (HNF-3) and the Drosophila homeotic gene fork head proteins are prototypes of an extensive family of cell-specific transcription factors that share homology in the winged helix DNA-binding domain. One of these mammalian family members, HNF-3/fork head homolog-4 (HFH-4), was isolated by PCR amplification of rodent brain cDNA and exhibits abundant expression in the adult bronchiolar epithelium. In this study, we performed in situ hybridization of stage-specific mouse embryos and report on a novel expression pattern of the HFH-4 gene in both the presumptive and differentiated choroid plexus epithelium, which is responsible for the synthesis and secretion of cerebrospinal fluid (CSF) proteins. We also showed that HFH-4 is a potent transcriptional activator in cotransfection assays and defined several protein sequences important for HFH-4 transcriptional activity. We used in vitro DNA-binding site selection with recombinant HFH-4 protein and determined that the HFH-4 protein recognizes the DNA consensus sequences HWDTGTTTGTTTA or KTTTGTTGTTKTW (where H is not G, W is A or T, D is not C, and K is G or T). We used this HFH-4 consensus to identify potential HFH-4 target genes in the choroid plexus epithelium and demonstrated that these promoter sequences bind to recombinant HFH-4 protein in electrophoretic mobility shift assays. Recombinant HFH-4 formed specific protein-DNA complexes with the promoter regions of the human prothrombin, beta amyloid precursor protein, alpha1-antichymotrypsin, cystic fibrosis transmembrane conductance regulator and rodent alpha2-macroglobulin, growth hormone receptors, and insulin-like growth factor II genes. Furthermore, we identified putative HFH-4 target genes in the bronchiolar epithelium including the clara cell secretory protein gene and the HNF-3alpha gene, a winged helix family member involved in the transcriptional regulation of genes in the bronchiolar epithelium. In support of these binding studies, cotransfection assays show that HFH-4 potentiates expression of the HNF-3alpha and clara cell secretory protein promoter regions.