Although recombinant adenoviruses are attractive vectors for gene transfer to airway epithelia, they have proven to be relatively inefficient. To investigate the mechanisms of adenovirus-mediated gene transfer to airway epithelia, we examined the role of adenovirus fiber and penton base, the two proteins involved in attachment to and entry of virus into the cell. We used human airway epithelia grown under conditions that allow differentiation and development of a ciliated apical surface that closely resembles the in vivo condition. We found that addition of fiber protein inhibited virus binding and vector-mediated gene transfer to immature airway epithelia, as well as to primary cultures of rat hepatocytes and HeLa cells. However, fiber protein had no effect on vector binding and gene transfer to ciliated airway epithelia. We obtained similar results with addition of penton base protein: the protein inhibited gene transfer to immature epithelia, whereas there was no effect with ciliated epithelia. Moreover, infection was not attenuated with an adenovirus containing a mutation in penton base that prevents the interaction with cell surface integrins. These data suggest that the receptors required for efficient infection by adenovirus are either not present or not available on the apical surface of ciliated human airway epithelia. The results explain the reason for inefficient gene transfer and suggest approaches for improvement.