The present study evaluates the hypothesis that delayed-type hypersensitivity (DTH) contributes to the inflammatory reaction observed when high-dose adenoviral (Ad) vectors are administered to a previously immunized animal. Immunocompetent C57BL/6 mice immunized intraperitoneally with 10(9) pfu AdCMV.Null [an E1-, E3- Ad vector with a cytomegalovirus (CMV) promoter but no transgene] and challenged intradermally to the footpad with the same vector demonstrated significant footpad swelling 24 hr after challenge with 10(9) pfu, but not with a lower dose. Footpad histology revealed a mononuclear-granulocytic cellular infiltrate typical of that seen in DTH. Evaluation of the same doses of vector in immunodeficient mice nu/nu and RAG-2- on the C57BL/6 background, and nu/nu and severe combined immunodeficiency (SCID) on the BALB/c background demonstrated suppression of footpad swelling. However, the footpad response remained intact in beta 2-microglobulin deficient (beta 2-m-) mice, suggesting minimal or no role of major histocompatibility complex (MHC) class I-mediated mechanisms for the region of localized inflammation. Challenge with an Ad expressing the interleukin-2 cDNA to immunized C57BL/6 mice demonstrated augmented footpad swelling response. Finally, pretreatment with cyclosporin resulted in a 69% inhibition of the response compared to controls, whereas other immunosuppressants (cyclophosphamide, methotrexate, and hydrocortisone) had no inhibitory effect. These findings provide further insight into the dynamic interplay of immune processes ultimately leading to inflammation when high-dose Ad vectors are administered to a target organ.