Human adenovirus type 5 can be used as a vector to elicit immune responses to antigens expressed from heterologous DNA sequences incorporated into the viral genome, for example in mice immunized intraperitoneally. We have used a recombinant adenovirus which expresses the p559(gag) antigen of simian immunodeficiency virus to evaluate the nature and longevity of the response elicited when administered to mice by alternative routes which translate more readily to larger animals and man. In C57Bl/6 mice immunized orally with a single dose of virus, a majority of the animals which showed evidence of responding to the immunogen by producing an anti-adenovirus response also produced a plasma antibody response to Gag which persisted for more than 1 year and a Gag-specific cytotoxic T cell response that could be detected for at least 6 months. In a minority of similarly immunized responding animals, only a cytotoxic response to Gag was observed although both humoral and cellular responses to adenovirus antigens were seen; intranasal immunization produced a Gag-specific response similar to this latter pattern. These findings suggest that delivery of adenovirus recombinants orally or intranasally may be a useful strategy for eliciting long-term cytotoxic T cell memory responses in splenocytes to candidate vaccine antigens.