We have characterized the ability of adeno-associated virus (AAV) serotypes 1-9 in addition to nineteen novel vectors isolated from various tissues, to transduce mouse and human ciliated airway epithelium (HAE). Vectors expressing alpha-1-antitrypsin (AAT) and beta-galactosidase were co-instilled into the mouse lung. Of all the vectors tested rh.64R1, AAV5 and AAV6 were the most efficient. The high transduction observed in mouse was reproduced in HAE cell cultures for both rh.64R1 and AAV6 but not for AAV5. Since AAV6 was the most efficient vector in mouse and HAE we also tested the transduction efficiencies of the AAV6 singleton vectors (i.e., AAV6 variants with targeted mutations) in these models. Of these, AAV6.2 transduced mouse airway epithelium and HAE with greater efficiency than all other AAV vectors tested. We demonstrated that AAV6.2 exhibits improved transduction efficiency compared to previously reported AAVs in mouse airways and in culture models of human airway epithelium and that this vector requires further development for preclinical and clinical testing.