Effective gene transfer and expression following repetitive administration of adenoviral (Ad) vectors in experimental animals is limited by anti-Ad neutralizing antibodies. Knowing that anti-Ad humoral immunity is serotype-specific, we hypothesized that anti-Ad neutralizing immunity could be circumvented using Ad vectors of different serotypes (Ad2, Ad5) within the same subgroup (C) to transfer and express β-glucuronidase (βglu) in the lung. Sprague-Dawley rats received an intratracheal administration of either Ad2βglu or Ad5βglu, and, 14 days later, repeat administration of either the same vector or a vector of a different serotype. Analysis of serum and bronchoalveolar lavage fluid following initial vector administration demonstrated systemic and local serotype-specific neutralizing antibodies. For both the Ad2 and Ad5 vectors, βglu expression 24 hr following the second administration of the same serotype was <30% of that of naive animals. In contrast, βglu expression 24 hr following second administration of a different serotype Ad vector was similar to expression at 24 hr of naive animals receiving a single administration (Ad5βglu followed by Ad2βglu, as well as Ad2βglu followed by Ad5βglu; p > 0.2 both comparisons). Although the alternative serotype bypassed anti-Ad neutralizing immunity, persistence of expression was reduced compared to that following administration to naive animals. Compatible with this observation, systemic administration of the same vectors to C57B1/6 mice demonstrated induction of cytotoxic T lymphocytes directed against the βglu transgene, as well as products of the Ad genome. Interestingly, intratracheal administration of vectors with different serotypes and different transgenes to rats resulted in longer expression (but still not normalized) compared to that achieved with vectors of different serotypes but the same transgene. These observations demonstrate that alternate use of Ad vectors from different serotypes within the same subgroup can circumvent anti-Ad humoral immunity to permit effective gene transfer after repeat administration, although the chronicity of expression is limited, likely by cellular immune processes directed against both the transgene and viral gene products expressed by the vector.