Lentiviral vectors have been tested as vaccination vectors in anti-tumoral and anti-viral models. They efficiently transduce dendritic cells and stimulate strong T-cell responses against the encoded antigen. However, their capacity to stimulate a cytotoxic T-lymphocyte (CTL) response against several antigens has not been evaluated. Broad anti-human immunodeficiency virus 1 (HIV-1) T-cell immune responses are important for the control of HIV replication. We evaluated the potential of polyepitope-encoding lentiviral vectors to induce broad anti-HIV CTL responses. We constructed two lentiviral vectors coding for an HLA-A2- or HLA-B7-restricted polyepitope and evaluated their immunogenicity by direct injection of vector particles in HLA-A2 or HLA-B7 transgenic mice. In vitro cytotoxicity assays showed that a single immunization induces a strong, diversified, and long-lasting CTL response in both mouse models. CTL responses were directed against all 13 epitopes in the HLA-A2 system and 8 out of 12 in the HLA-B7 system. A second immunization augmented the number of responding mice in the HLA-A2 system but not in the HLA-B7 system. HLA-B7-immunized mice mounted strong interferon-gamma (IFN-gamma)-secreting T-cell responses against a majority of the epitopes and lysed peptide-loaded target cells in vivo. CTL responses in HLA-B7 mice were only partially dependent on CD4 T-cell help. This work underlines the potential of lentiviral vectors as candidates for therapeutic vaccination against acquired immunodeficiency syndrome.