Genetic vaccination with adenoviral (Ad) gene transfer vectors requires transduction of professional antigen-presenting cells. However, because the natural Ad receptors are expressed on many cell types, the Ad vectors currently in use are characterized by high promiscuity. In fact, the majority of injected Ad vector particles are likely to transduce non-target cells. We have analyzed various sizes of polyethylene glycol (PEG) molecules for vector particle detargeting, and our data provide evidence that the size of the PEG determines detargeting efficiency. With the use of appropriately large PEG molecules, vector particles were detargeted from muscle after local delivery and from liver after systemic delivery in mouse models. Surprisingly, fully detargeted PEGylated Ad vectors still induced strong cellular and humoral immune responses to vector-encoded transgene products. Also, injection of PEGylated and non-PEGylated vector particles resulted in similar kinetics of transgene product-specific cytotoxic immune responses, thereby suggesting that the same cell types were involved in their induction. Furthermore, we showed that PEGylated vectors evade neutralizing anti-Ad antibodies in vivo. This feature might help circumvent the recognized limitation imposed by the widespread occurrence of anti-Ad immunity in the human population. We suggest that PEGylated Ad particles with significantly reduced promiscuity may qualify as a novel and safe vector format for genetic vaccination.