Expression of antigens in dendritic cells (DC) can stimulate protective immunity against both viral infection and tumor growth, making them important targets for gene therapy. In-vitro-generated DC are commonly used in gene delivery studies with the assumption that the results will correlate with in vivo activity. Adenovirus Type 5 (Ad5) vectors have been widely used with DC, but these cells lack the primary receptor (CAR) used by Ad5 and are poorly infected. We investigated the use of Ad5 vector particles pseudotyped with fibers from other Ad serotypes in DC targeting. Several fiber proteins, including those from Ad16 (Subgroup B) and Ad37 (Subgroup D), conferred dramatically increased in vitro infection. Surprisingly, neither dendritic cell infection nor the immune response to an Ad-delivered antigen was improved when the modified viruses were tested in vivo. These results underscore the importance of using appropriate animal models in gene delivery studies.