The production of an efficacious HIV-1 vaccine capable of inducing the humoral and cellular arms of the immune system continues to remain a major challenge in AIDS research. Dendritic cells (DC) are an attractive target for vaccine delivery because vaccine antigens presented to DC can potentially activate effector cells involved in immune defense. Adenovirus (Ad) has been widely used as a vector to express foreign proteins in a variety of cell types and tissues. However, this broad tropism represents a drawback when selective gene delivery is desired. In particular, inefficient gene delivery to antigen presenting cells (APC) is attributed to the absence of the Coxsackie and Adenovirus receptor (CAR). Based on the hypothesis that Ad vectors with improved infectivity of APC may be able to promote an enhanced immune response against a vectored antigen, our objective is to selectively deliver Ad vectors to APC by directing virus to the high affinity Fc-gamma Receptor I (FcgRI), constitutively expressed on DC. To this end, we constructed a bi-specific targeting molecule, H22sCAR, consisting of the extracellular domain of CAR (sCAR), a bacteriophage T4 fibritin trimerization domain, and a single-chain antibody molecule (H22) specific for the human FcgRI receptor. H22sCAR was produced in a baculovirus expression system, and was demonstrated to bind both the Ad fiber knob protein and cellular FcgRI. Ad vectors pre-complexed with H22sCAR enhanced the delivery of GFP and luciferase reporter genes to FcgRI-expressing cell lines, and to monocyte-derived DC, when compared with Ad alone or Ad targeted with an irrelevant control protein (Her2sCAR). These data support the hypothesis that FcgRI-targeted Ad vectors may enhance the delivery of target antigen to APC, and lend support to future studies on the use of FcgRI-targeted adenovirus vectors for HIV-1 vaccine development.