We previously demonstrated that systemic administration of adenovirus serotype 35 (Ad35) vectors to mice does not mediate efficient transduction in organs, probably because expression of the mouse analog of the subgroup B Ad receptor, human CD46 (membrane cofactor protein), is limited to the testis. Here, we describe the in vitro and in vivo transduction characteristics of Ad35 vectors by using homozygous and hemizygous human CD46-transgenic (CD46TG) mice, which ubiquitously express human CD46. An Ad35 vector more efficiently transduced the primary dendritic cells and macrophages prepared from CD46TG mice than those from wild-type mice. In vivo transduction experiments demonstrated that CD46TG mice are more susceptible to Ad35 vector-mediated in vivo transduction than are wild-type mice. In particular, homozygous CD46TG mice, which express higher levels of CD46 in the organs than hemizygous CD46TG mice, tend to exhibit higher transduction efficiencies after intraperitoneal administration than hemizygous CD46TG mice. Intraperitoneal administration of Ad35 vectors resulted in efficient transduction into the mesothelial cells of the peritoneal organs in homozygous CD46TG mice. These results indicate that an Ad35 vector recognizes human CD46 as a cellular receptor in CD46TG mice. However, the in vivo transduction efficiencies of Ad35 vectors in CD46TG mice are much lower than those of conventional Ad5 vectors in wild-type mice.