While 51 human adenoviral serotypes have been identified to date, the vast majority of adenoviral vectors designed for gene transfer have been generated in the adenovirus serotype 5 (Ad5) backbone. Viral infections caused by Ad5 are endemic in most human populations and the majority of humans carry preexisting humoral and/or cellular immunity to Ad5 which may severely limit the use of Ad5-based vectors for gene therapy applications. To circumvent this preexisting Ad5 immunity, we have identified Ad35 as an alternative adenoviral serotype to which the majority of humans do not have neutralizing antibodies. Importantly, Ad35 can be grown to high titers with a low particle-to-PFU ratio. As a prerequisite for the development of Ad35 for use as a gene transfer vector, a genome organization map was constructed using the available Ad35 sequence information, and E1a-deficient Ad35 vectors encoding marker genes were generated. Ad35 biodistribution in mice was assessed following intravenous administration and compared with that of Ad5. Extremely low levels of Ad35 were detected in all organs evaluated, including liver, lung, spleen, and bone marrow, while Ad5 displayed high transduction of these organs. Due to the lack of Ad35 liver tropism, minimal hepatotoxicity was observed in mice treated with Ad35. Furthermore, the half-life of Ad35 in mouse blood was found to be two to three times longer than that of Ad5. These data suggest that either mice do not express the Ad35 cell surface receptor or that Ad35 does not efficiently transduce mouse cells in vivo following systemic delivery. Therefore, to begin to elucidate the Ad35 cell entry mechanisms, in vitro competition studies were performed. These data demonstrated that Ad35 cell entry is CAR independent, and may involve protein(s) expressed on most human cells.