Cystic fibrosis (CF) is a life‐threatening genetic disease caused by mutations in the CF transmembrane conductance regulator gene. It is the most common genetic disease among Caucasians. There is an urgent need to create more therapies for CF since current drugs are not effective in treating all the mutations that cause this disease. Furthermore, gene therapy is a potential treatment for CF airway disease. Because of this, various viral‐based gene transfer vectors have been evaluated for their efficiency in correcting the CF airway disease phenotype. This project aimed to compare side‐by‐side the differences in the transduction efficiency and targeting of specific cell types of adenovirus (Ad)‐based and adeno‐associated virus (AAV)‐based vectors in different models of the airway. Different serotypes of AAV, namely 1, 2, 5, 6, and Ad5‐based vectors, expressing green fluorescent protein (GFP), firefly luciferase (ffLuc) or ß‐galactosidase (LacZ) were assessed in in vitro and in vivo gene transfer studies. Madin‐Darby Canine Kidney (MDCK) cells were grown submerged on plastic, and human airway epithelial (HAE) cells were grown on transwells and fed only from the basolateral side to attempt to model the human conducting airways. The viral vectors expressing GFP were tested at doses of 102–103 genome copies (GC) for AAV or plaque‐forming unit (pfu) for Ad, and the level of transduction was assessed at different time points. The data demonstrates that in MDCK and HAE cells, the highest transduction was achieved by the Ad5 vector followed by the AAV2 vector, while AAV6 had the lowest transduction. Additionally, as examined in HAE cells, the age of the culture impacts transduction efficiency. In parallel, AAV vectors 1, 2, 5, 6 (at a dose of 8×1010 – 3×1011 GC) and Ad5 vector (at a dose of 1×1011 pfu) expressing ffLuc or LacZ were intranasally delivered to mice to assess the transduction efficiency in the epithelium of both the nose and lungs. AAV6 was the most efficient at transducing the cells of the nasal epithelium of mice and AAV5, at transducing lung cells. AAV2 did not transduce airway cells in mice. In conclusion, we confirmed that the viral vectors have different transduction profiles in different models of airway epithelium. More airway models should be studied to better understand which are more useful for preclinical assessments of CF gene therapeutics.Support or Funding InformationThis project was supported by the National Heart, Lung, and Blood Institute (R25‐HL084665‐14).