Complexes of DNA and cationic lipid offer potential advantages for gene transfer to airway epithelia. However, we found that application of DNA–lipid (DMRIE–DOPE) complexes to primary cultures of human ciliated airway epithelia or explants of rabbit trachea generated only low levels of gene transfer. In contrast, when we applied the DNA–lipid to immature human epithelia shortly after seeding, transgene expression was substantially higher. We identified two barriers that limit gene transfer. First, uptake of the DNA–lipid complexes into airway cells across the apical membrane decreased rapidly with time after seeding and paralleled the decrease in transgene expression. Second, cell division decreased with time after seeding, and we found that cells in mitosis (labeled with BrdU) were much more likely to express transgene than BrdU-negative cells. These data suggest that the entry step across the apical membrane and the low rate of cell division are important barriers for cationic lipid-mediated gene transfer to airway epithelia. Attempts to modify these two processes may yield improvements in the efficiency of gene transfer to the airways in cystic fibrosis.