Intratracheal instillation of perfluorochemical (PFC) liquids enhances lung epithelial transgene expression by improved vector propulsion throughout lung airways. We now demonstrate that PFC liquids also facilitate gene transfer by increasing transepithelial permeability. Apical application of PFC liquid to well-differentiated human airway epithelial cells resulted in a transient decrease in transepithelial resistance peaking approximately 2 h after PFC liquid administration and returning to normal approximately 24 h later. The permeability change was sufficient to enhance access of apically applied 100-nm latex beads and adenoviral vectors to the basolateral side of the culture. Adenovirus-mediated gene expression was concurrently enhanced. Following intratracheal instillation of PFC liquid into mouse lungs, tight junction permeability, as assessed by electron microscopic evaluation of lanthanum deposition, was increased with peak effect between 6 h and 1 day after instillation. Importantly, alveolar-capillary permeability remained unchanged with the treatment. Administration of PFC liquid 6 h or 1 day, but not 3 days, prior to instillation of a recombinant adenovirus vector enhanced gene expression comparable to that observed with concurrent administration of PFC liquid and vector. We conclude that transient increase in epithelial permeability after PFC liquid administration contributes to the enhancement of adenovirus vector-mediated gene expression in lung epithelium.