Human immunodeficiency virus type 1 (HIV-1) infection is characterized by the rapid onset of intestinal T-cell depletion that initiates the progression to AIDS. The induction of protective immunity in the intestinal mucosa therefore represents a potentially desirable feature of a preventive AIDS vaccine. In this study, we have evaluated the ability of an enteric adenovirus, recombinant adenovirus 41 (rAd41), to elicit intestinal and systemic immune responses by different immunization routes, alone or in combination with rAd5. rAd41 expressing HIV envelope (Env) protein induced cellular immune responses comparable to those of rAd5-based vectors after either a single intramuscular injection or a DNA prime/rAd boost. Oral priming with rAd41-Env followed by intramuscular boosting with rAd5-Env stimulated a more potent CD8(+) T-cell response in the small intestine than the other immunization regimens. Furthermore, the direct injection of rAd41-Env into ileum together with intramuscular rAd5-Env boosting increased Env-specific cellular immunity markedly in mucosal as well as systemic compartments. These data demonstrate that heterologous rAd41 oral or ileal priming with rAd5 intramuscular boosting elicits enhanced intestinal mucosal cellular immunity and that oral or ileal vector delivery for primary immunization facilitates the generation of mucosal immunity.