Treatment of cystic fibrosis by gene therapy will require the development of vectors capable of efficient and safe transfer of a functional cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to airway epithelia. To achieve this goal, replication-deficient (E1-) adenoviruses (Ad) are promising vectors. We have previously demonstrated efficient CFTR gene delivery to the airways of cotton rats and rhesus monkeys using a replication-deficient adenovirus, Ad-CFTR. Here, we have investigated an important safety issue, the interaction between the vector and wild-type virus which can provide the missing E1 function in trans. We show that Ad5 can mobilize the defective Ad-CFTR genome in vitro and in cotton rats. However, the extent of the complementation in vivo by wild-type virus is limited because no additional spreading or shedding of Ad-CFTR to trachea, lungs, and stools is elicited. To attenuate Ad-CFTR further, a mutation was introduced in the cis-acting regulatory sequences that control the encapsidation of the viral genome. We demonstrate that when cells are coinfected with wild-type virus and the new attenuated vector, the viral DNA containing the natural encapsidation sequences is preferentially packaged, leading to a rapid dilution of the recombinant virus.