Over one thousand mutations have been identified to date in the cystic fibrosis transmembrane conductance regulator gene (CFTR); however, about 5–10% of mutations remain undefined. It is likely that some of these undefined mutations occur within regulatory elements for the CFTR gene. Tissue-specific regulatory elements for CFTR are located outside the basal promoter region and may be associated with DNase I hypersensitive sites (DHS). We previously described a DHS at +15.6 kb 3′ to the CFTR gene, which showed tissue specificity in vivo and was evaluated as a candidate regulatory element for CFTR. Polymorphisms in regulatory elements may have a significant effect on their activity and hence on the levels of gene expression. Two C→T polymorphisms were identified within the +15.6 kb region that occurred on both cystic fibrosis (CF) and non-CF alleles. Both of the polymorphisms altered DNA-protein binding, as shown by electrophoretic mobility shift assays (EMSA). These changes in transcription factor binding at a putative regulatory region could influence CFTR gene expression.