We evaluated the effect of immunization with dendritic cells (DCs) pulsed with alpha-galactosylceramide (alphaGalCer) and listeriolysin O (LLO) 91-99 peptide, a dominant cytotoxic T lymphocyte (CTL) epitope of Listeria monocytogenes by observing the responses of specific CD8(+) T cells and in vivo CTL activity. DCs were pulsed with various combinations of alphaGalCer and LLO91-99 peptide and administered to BALB/c mice. Immunization with DCs pulsed with alphaGalCer and LLO91-99 at priming phase and with DCs pulsed with LLO91-99 alone at boosting phase induced stronger in vivo CTL activity, reduced the bacterial load in spleens of Listeria-challenged mice and augmented CD62L(+) CD8(+) central memory T cells compared with other immunization protocols. The blockade of interferon-gamma (IFN-gamma) at boosting phase reversed the induction of CD8(+) central memory T cells and reduced the bacterial load in spleens of Listeria-challenged mice immunized with DCs pulsed with alphaGalCer and LLO91-99 at both phases, suggesting that alphaGalCer at boosting phase has deleterious effects through IFN-gamma production. These results indicate that immunization with DCs pulsed with CTL epitope peptide together with alphaGalCer at priming phase, but not at boosting phase, is feasible for eliciting a specific CTL activity and protective immunity against infection of intracellular bacteria.