Efforts have been made to deliver transgenes to the airway epithelia of laboratory animals and humans to develop gene therapy for cystic fibrosis. These investigations have been disappointing due to combinations of transient and low-level gene expression, acute toxicity, and inflammation. We have developed new helper-dependent adenoviral vectors to deliver an epithelial cell-specific keratin 18 expression cassette driving the beta-galactosidase (beta-gal) or human alpha-fetoprotein (AFP) reporter genes. Following intranasal administration to mice, we found that the reporter genes were widely expressed in airway epithelial and submucosal cells, and secreted human AFP was also detectable in serum. In contrast to a first-generation adenoviral vector, inflammation was negligible at doses providing efficient transduction, and expression lasted longer than typically reported-up to 28 days with beta-gal and up to 15 weeks with human AFP. These results suggest that delivery to the airway of helper-dependent adenoviral vectors utilizing a tissue-specific promoter could be a significant advance in the development of gene therapy for cystic fibrosis.