A mouse model of chronic Pseudomonas-induced bronchopulmonary inflammation that mimics chronic cystic fibrosis (CF) lung disease was employed to determine whether this inflammatory milieu influences immune responses to adenoviral vectors. Pseudomonas-infected and control mice were inoculated intranasally with a second-generation type 2 adenovirus (Ad2) vector (Ad2/betagal-2). After 3 weeks, serum and airway Ad2-specific antibodies and Ad2 vector-directed, cytotoxic T-lymphocyte (CTL) activity in splenocytes were measured. No differences in humoral immunity were observed between Pseudomonas-infected mice and controls. However, there was a two- to three-fold increase in Ad-specific CTL activity in the Pseudomonas-infected mice compared to control mice. MHC class I-dependent antigen presentation by antigen-presenting cells (APC) from lungs of Pseudomonas-infected mice was also significantly increased compared to APC from control mice, suggesting a mechanism that may contribute to increased Ad-specific CD8+ CTL responses. It was concluded that Ad-specific CTL activity is enhanced in the setting of pre-existing chronic Pseudomonas-induced lung inflammation similar to CF lung disease, and that increased antigen presentation via MHC class I in this setting may be one underlying mechanism. These findings underscore the importance of considering the influence of the disease milieu when evaluating modes of gene therapy for such diseases in animal models.