In contrast to commonly used serotype 5-based adenovirus (Ad) vectors, Ad's containing fibers derived from B-group serotype 35 (Ad5/35) efficiently transduce human DCs ex vivo and appear to target antigen-presenting cells after intravenous injection into baboons. Based on this, Ad5/35 vectors could be valuable tools for immunotherapy and vaccination. On the other hand, a number of studies indicate that signaling through the B-group Ad receptor, CD46, can cause tolerance or immunosuppression. Since mice do not express CD46 in a human-like pattern, we studied the in vivo properties of Ad5/35 in transgenic mice that express CD46 in a pattern and at a level similar to those of humans. Hypersensitivity assays and analyses of frequencies of regulatory T cells and T cell responses did not indicate that Ad5/35 injection exerts detrimental effects on the host's immune system. An Ad5/35 vector expressing a model antigen was able to trigger a strong T cell response against the test antigen after intramuscular injection. Overall, compared to Ad5 vectors, Ad5/35 vectors had a better safety profile, reflected by lower serum levels of proinflammatory cytokines.