The tissue-specific regulation of the cystic fibrosis transmembrane conductance regulator gene (CFTR) is coordinated by intronic and extragenic cis-acting elements that influence its transcriptional activity. The promoter apparently lacks sequences to drive cell type-specific expression. We previously identified a number of intronic elements that were associated with DNase I hypersensitive sites (DHS) and bound the hepatocyte nuclear factor 1 (HNF1) transcription factor. Moreover, we demonstrated the likely involvement of HNF1 in the regulation of CFTR expression in vivo. Here we investigate DHS in introns 16 and 17a of the CFTR gene, which are evident in intestinal and pancreatic cell lines, and determine the transcription factors that interact with these sites. Of particular interest were factors known to interact with HNF1 in coordinated expression of genes in the gastrointestinal tract. We demonstrate that though sequences within these DHS bind HNF1, CDX2, and PBX1 in vitro, only PBX1 show a robust in vivo interaction. These data contribute to our understanding of the complexity of cell-type-specific CFTR regulatory mechanisms.