We have previously shown that vaccines expressing virus-derived cytotoxic-T-lymphocyte (CTL) epitopes as short minigenes can confer effective protection against virus challenges, and here we extend these studies to the bacterium Listeria monocytogenes. Host defense against this important human pathogen appears largely T cell mediated, and a nonamer CTL epitope from the listeriolysin O (LLO) protein has been identified in BALB/c mice. We have synthesized this nonamer as a minigene, expressed it in a recombinant vaccinia virus (VV-list), and used this to immunize mice. Memory CTLs cultured from VV-list-immunized mice specifically lyse target cells pulsed with a nonamer peptide identified at LLO amino acid residues 91 to 99. Four weeks postimmunization, mice were challenged with L. monocytogenes. By day 6 following challenge with a sublethal dose of L. monocytogenes, mice immunized with VV-list showed a approximately 2,000- to 6,000-fold reduction in bacteria CFU in the spleen and liver. At this time point, with control mice, bacterial were readily detectable by Gram stain of the liver but were undetectable in the VV-list-immunized animals. Additionally, when a normally lethal dose of bacteria was given, death was delayed in VV-list-immunized animals. This study has demonstrated that a single immunization with a recombinant vaccinia virus bearing only nine amino acids from a bacterial pathogen can induce specific CTLs able to confer partial protection against bacterial challenge.