Adenoviral vectors deleted of E1 are attractive vehicles for in vivo gene therapy because efficient gene transfer can be achieved. Immune responses to the vector and vector-transduced cells lead to destruction of target cells, inflammation and difficulties with vector readministration. Immune effectors have been identified as CD8+ cytotoxic T lymphocytes, which destroy vector-transduced cells, as well as B cells which secrete neutralizing antibodies and block repeated gene transfer. The central role of CD4+ T cells in the activation of both of these effector functions has focused immunosuppressive strategies towards blockade of costimulatory molecules. We describe in this study a strategy which aims to inhibit CD4+ T cell activation by transiently administering an inhibitor of the CD28/B7 pathway, ie CTLA4Ig, at the time an E1-deleted adenovirus is administered to liver or lung. In lung, CTLA4Ig treatment significantly blocked the formation of neutralizing antibodies, allowing efficient readministration of virus, whereas transgene expression was only moderately prolonged. In contrast, CTLA4Ig did not suppress neutralizing antibody formation in the context of liver gene therapy, but resulted in more stable gene expression. In vitro assays revealed suppression of T cell activation in either organ. This observation suggests that transient inhibition of the CD28/B7 pathway at the time of virus instillation can partially interfere with both arms of the immune response to adenovirus-mediated gene transfer circumventing the need for chronic immune suppression.