Animal studies have suggested that the clinical usefulness of recombinant adenoviruses (Ad) as vectors for therapeutic gene delivery may be limited by their immunogenicity. Neutralizing antibodies elicited by capsid proteins reduce the efficiency of vector readministration whereas cytotoxic T lymphocytes (CTLs) directed against viral proteins and/or immunogenic transgene products expressed by transfected cells have the potential to limit persistence of expression. In this study, transient administration of the novel immunosuppressant deoxyspergualin (DSG) was found to inhibit the development of both humoral and cell-mediated immune responses against Ad vector delivered intranasally. DSG treatment of primed mice previously exposed to wild-type Ad impaired the development of antibodies in response to a secondary and even tertiary challenge with Ad vector. As a result, improved gene transfer was obtained upon subsequent administration of a beta-galactosidase (beta-Gal)-encoding Ad vector. Short-term administration of DSG also depressed the activation of CD4+ and CD8+ T lymphocytes as assessed by measurement of antigen-specific proliferation and CTL activity, respectively. The marked suppression of CTL activity against Ad vector in DSG-treated mice correlated with improved persistence of transgene expression in the lung.