Recombinant adenoviral vectors (AdV) have been used experimentally as vaccines to present antigenic transgenes in vivo. However, administration of first-generation vectors (FG-AdV) is often limited by their induction of antiviral immunity. To address this limitation, helper-dependent vectors (HD-AdV) were developed that lack viral coding regions. While the administration of HD-AdV results in long-term gene expression in vivo, their utility as immunogens has never been examined. Direct vaccination with 10(8) blue-forming units (BFU) of HD-AdV injected into C57BL/6 mice lead to superior transgene-specific CTL and antibody responses when compared to the same amount of a FG-AdV. The antibody responses to viral antigens were high in response to both the vectors. As a mechanism to reduce viral exposure, dendritic cells (DC) were transduced with HD-AdV in vitro and then used as a cell-based vaccine. DC transduced with HD-AdV expressed higher levels of transgene-specific mRNA and up to 1200-fold higher levels of transgene protein than did DC transduced with a FG-AdV. In addition, HD-AdV-transduced DC stimulated superior transgene-specific CTL responses when administered in vivo, an effect that was further enhanced by maturing the DC with LPS prior to administration. In contrast to direct immunization with HD-AdV, vaccination with HD-AdV-transduced DC was associated with limited antibody responses against the AdV. We conclude that HD-AdV stimulates superior transgene-specific immune responses when compared to a FG-AdV, and that immunization with a DC-based vaccine maintains this efficacy while limiting antiviral reactivity.