Adenovirus-mediated gene transfer experiments have demonstrated an exceptional efficiency of virus uptake and gene expression in a variety of in vivo models. Unfortunately, the efficiency of gene delivery is not accompanied by long-term gene expression. Maximal gene expression peaks during the first week of infection followed by a rapid decline to near baseline levels within several weeks. Data from several laboratories implicate host cellular and humoral immune responses as being responsible for the limited duration of expression and for the inability to successfully readminister a gene using adenovirus vectors. In this study we have examined two strategies which, independently or in combination, circumvent aspects of the host immune response against adenovirus-mediated gene therapy. The first strategy explores induction of immune tolerance in the experimental host as a method to increase the duration of gene expression and as a method to allow readministration of adenovirus expression vectors. Our second strategy is directed at the need to readminister adenoviral vectors to immune competent adult animals. We have demonstrated that a sequential exposure of rats to at least two other adenovirus serotypes does not compromise our ability to successfully administer an Ad5-based CAT expression vector. The characterization of serotype-specific neutralizing response indicates that the construction and use of Ad expression vectors from different serotypes will facilitate a useful adenovirus-based strategy allowing multiple administrations of a target gene.