The utility of adenoviral vectors is limited by immune responses to adenoviral antigens. We sought to develop immune-competent mice in which the immune response to adenoviral antigens was selectively absent. To do so, we generated mice that were transgenic for a replication-defective vector. Adenoviral antigens might be seen as self-antigens by these mice, and the mice could exhibit immunologic tolerance after postnatal exposure to adenoviral vectors. In addition, characterization of these mice could reveal potential consequences of germline transmission of an adenoviral vector, as might occur in a gene therapy trial. Injection of a "null" (not containing a transgene) E1, E3-deleted vector genome into mouse zygotes yielded five founders that were capable of transmitting the vector genome. Among offspring of these mice, transgenic pups were significantly underrepresented: 108 of 255 pups (42%) were transgenic (P<0.02 versus expected frequency of 50%). Postnatal transgenic mice, however, had no apparent abnormalities. Persistence of an adenoviral vector after intravenous injection was equivalent in livers of transgenic mice and their nontransgenic littermates. Transgenic and nontransgenic mice also had equivalent humoral and cellular immune responses to adenoviral vector injection. Mice that are transgenic for an E1, E3-deleted adenoviral genome can be easily generated; however, they are not tolerant of adenovirus. Moreover, germline transmission of an adenoviral vector genome does not prevent generation of a robust immune response after exposure to adenoviral antigens.