CTL responses are known to be important for the control of HIV and SIV infections. Such responses are targeted against various components of these viruses including regulatory proteins like Nef. The SIV(mac251)nef gene was cloned in Mycobacterium bovis BCG under the control of P-AN, a promoter from Mycobacterium paratuberculosis. Nef was expressed as a fused polypeptide with ORF2, an open reading frame adjacent to P-AN. Mice inoculate with rBCG(SIV(mac251)nef) exhibited proliferative and CD8+ cytotoxic T-cell (CTL) responses against several Nef synthetic peptides. A mapping of the epitopes recognized by CTLs revealed that the central region of Nef is mainly involved in responses. This region had already been demonstrated to induce CTLs in experimentally SIV-infected macaques as well as in HIV-infected individuals. These results demonstrate the feasibility of constructing BCG vaccine strains expressing nef for eliciting cytotoxic responses.