Previous non-human primate studies have shown replication competent adenovirus (Ad) HIVenv/rev and SIVenv/rev recombinants to be promising vaccine candidates. To broaden induced immunity in rhesus macaques, an Ad type 5 host range (Ad5hr) mutant vector with an inserted SIV gag gene was added to the vaccine regimen. Immunity to the encoded SIV Env, Rev, and Gag gene products was evaluated following two immunizations with the same recombinants. The vaccines were administered intranasally plus orally via stomach tube at weeks 0 and 12. The recombinants replicated well in the upper respiratory tract but poorly in the gut, suggesting enteric-coated capsules might improve oral delivery to the intestine. SIV-specific cellular immunity was induced in all 16 immunized macaques. Fourteen exhibited positive interferon-gamma (IFN-gamma) ELISPOT responses, and nine, including two lacking IFN-gamma responses, exhibited SIV-specific T-cell proliferative activity. IFN-gamma secreting peripheral blood mononuclear cells (PBMCs) in response to SIV Gag, Env, and Rev peptides were induced in 73, 53, and 27% of macaques, respectively, and were boosted two- to four-fold by the second immunization. A persistent response to Gag was evident at least 10 weeks thereafter. p11C tetramer staining confirmed elicitation of SIV Gag-specific CD8+ T-cells in Mamu-A*01 macaques. Proliferative responses were more frequent after the second immunization, and binding antibody titers to SIV gp120 were significantly boosted by the immunization regimen. We conclude that a second administration of recombinants based in the same Ad5hr vector can effectively boost immunity to inserted gene products, obviating development of several recombinants in different Ad serotypes for multiple immunizations.