The lack of vectors for selective gene delivery to the intestine has hampered the development of gene therapy strategies for intestinal diseases. We hypothesized that chimeric adenoviruses of Ad5 (species C) displaying proteins of the naturally enteric Ad40 (species F) might hold the intestinal tropism of the species F and thus be useful for gene delivery to the intestine. As oral-fecal dissemination of enteric adenovirus must withstand the conditions encountered in the gastrointestinal tract, we studied the resistance of chimeric Ad5 carrying the short-fiber protein of Ad40 to acid milieu and proteases and found that the Ad40 short fiber confers resistance to inactivation in acidic conditions and that AdF/40S was further activated upon exposure to low pH. In contrast, the chimeric AdF/40S exhibited only a slightly higher protease resistance compared with Ad5 to proteases present in simulated gastric juice. Then, the biodistribution of different chimeric adenoviruses by oral, rectal, and intravenous routes was tested. Expression of reporter β-galactosidase was measured in extracts of 15 different organs 3 days after administration. Our results indicate that among the chimeric viruses, only intrarectally given AdF/40S infected the colon (preferentially enteroendocrine cells and macrophages) and to a lesser extent, the small intestine, whereas Ad5 infectivity was very poor in all tissues. Additional in vitro experiments showed improved infectivity of AdF/40S also in different human epithelial cell lines. Therefore, our results point at the chimeric adenovirus AdF/40S as an interesting vector for selective gene delivery to treat intestinal diseases. © 2013, Mary Ann Liebert, Inc.