Recombinant, replication-deficient adenovirus (Ad) vectors have been successfully used to transfer and express the normal human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA in vivo in the respiratory epithelium of experimental animals and humans with cystic fibrosis (CF). Since Ad-directed gene expression wanes over time, repeat administration is necessary to achieve an effective treatment for CF. A major hurdle to such a strategy is the possibility that anti-Ad humoral immunity may prevent gene expression in individuals with pre-existing anti-Ad immunity or following repeat administration. One strategy to circumvent such a problem would be alternating the use of Ad vectors belonging to different subgroups. Neutralizing antibodies developed with the administration of one Ad serotype do not cross-react with an Ad belonging to a second serotype in a manner that blocks infection and gene expression. To test this hypothesis, an immunizing dose of wild-type Ad5 (subgroup C), Ad4 (subgroup E), or Ad30 (subgroup D) was administered intratracheally to experimental animals, followed by an intratracheal administration of a replication-deficient subgroup C-derived vector coding for marker genes (chloramphenicol acetyl transferase or β-galactosidase) or for the normal human CFTR cDNA. As expected, studies with vectors coding for marker genes or for CFTR cDNA demonstrated that airway administration of a vector does not yield efficient gene transfer, if there has been prior recent airway administration of the same Ad subgroup. In contrast, effective expression from the second administration can be achieved with an adenovirus vector belonging to a subgroup different from the first adenovirus administered. These data support the paradigm of alternating Ad vectors derived from different subgroups as strategy to circumvent anti-Ad humoral immunity, thus permitting the use of Ad vectors as a means to treat the respiratory manifestations of CF.