First-generation adenovirus vectors will have limited application in gene therapy for chronic diseases because of destructive host immune responses. Important immune effectors include CD8+ T cells, which mediate target cell destruction and ablate transgene expression, and B cells, which produce neutralizing antibodies that block effective readministration of vector. Previous studies indicated that activation of CD4+ T cells by virus capsid proteins is necessary for full realization of effector function of CD8+ T cells and B cells. In this paper, we present a strategy for preventing CD4+ T-cell activation by an adenovirus vector delivered to mouse liver and lung tissues which is based on interfering with T-cell priming via CD40 ligand-CD40 interactions. Adenovirus transgene expression was stabilized in mice genetically deficient in CD40 ligand (CD40L), and neutralizing antibody to adenovirus did not develop, allowing efficient readministration of vector. A transient blockade of T-cell activation with an antibody to CD40L infused into the animal at the time of adenovirus vector-mediated gene transfer led to stabilization of transgene expression and diminished production of neutralizing antibody, allowing readministration of vector. In vitro T-cell assays suggested that a block in the primary activation of CD4+ T cells was responsible for the lack of B-cell- and cytotoxic-T-cell-dependent responses. This suggests a strategy for improving the potential of adenovirus vectors based on administration of an antibody to CD40L at the time of vector administration.