We developed an epithelium-specific, inducible cystic fibrosis transmembrane conductance regulator (CFTR) expression system. In this system we used a human cytokeratin 18 expression cassette to drive epithelium-specific expression of the reverse tetracycline transactivator (rtTA), which turns on CFTR expression from a Tet-inducible promoter in the presence of doxycycline. CFTR expression was monitored by reverse-transcription polymerase chain reaction, immunostaining, and Western blotting. We confirmed that protein expression was dose-dependent in double stable transfected cell lines, with no detectable protein in the absence of doxycycline. However, low levels of CFTR mRNA could be detected in the uninduced state. When clones capable of inducing high levels of CFTR expression were analyzed, we observed a decrease in cell proliferation, consistent with reports in other cell lines (NIH3T3 and BTS). We generated transgenic mice expressing rtTA from the K18 expression cassette and demonstrated that the system retained its tissue specificity for lacZ reporter expression in vivo. When mice were induced with doxycycline, high levels of expression were found in the trachea, upper bronchi, and submucosal glands. Therefore, this inducible system can improve our understanding of the role of CFTR in the lung and should help in the design of safe and effective CF therapies.