Adenovirus serotype 35 (Ad35) vectors have shown promise as effective gene delivery vehicles. However, the transduction profiles of Ad35 vectors in conventional mice allow only a limited estimation of transduction properties of these vectors, because the mouse analog of the subgroup B Ad receptor, CD46, is restricted to the testis. In order to assess the transduction properties of Ad35 vectors more completely, we performed transduction experiments using cynomolgus monkeys, which ubiquitously express CD46 in a pattern similar to that in humans. In vitro transduction experiments demonstrated that cultured cells from the cynomolgus monkey were efficiently transduced with Ad35 vectors. In contrast, after intravenous administration into live monkeys hardly any evidence of Ad35 vector–mediated transduction was found in any of the organs, although Ad35 vector genomes were detected in various organs. Less severe histopathological abnormalities were found in the Ad35 vector–infused monkeys than in the conventional Ad5 vector–injected monkeys. In the latter, serious tissue damage and inflammatory responses, such as hepatocyte necrosis and lymphatic hyperplasia in the colon, were induced. Both Ad35 and Ad5 vectors caused similar hematological changes (increase in CD3⁺ cells, and decrease in CD16⁺ cells and CD20⁺ cells) in peripheral blood cells. These results should provide valuable information for the clinical application of Ad35 vectors.