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Adenovirus (Ad)-mediated transduction of dendritic cells (DCs) is inefficient because of the lack of the primary Ad receptor, CAR. CD40 is a surface marker expressed by DCs that plays a crucial role in their maturation and subsequent stimulation of T cells. DC infection with Ad targeted to the CD40 results in increased gene transfer. Cells transduced with CD40-targeted Ad5-SV40-TAg vector showed increased expression of transgene and expression of costimulatory molecules at 48 hours post-infection compared to cells transduced with untargeted Ad5-SV40-TAg vector. We demonstrated that CD40-targeted gene transfer promotes DC maturation with induction of a complex signaling cascade accompanied by characteristic changes in cytokine production. These results demonstrate that DCs can be successfully transduced using a CD40 targeted adenoviral vector and that transduced DCs show activation.