The immunologic mechanism of protective immunity to the intracellular parasite Listeria monocytogenes (Lm) is not well understood, however, antilisterial immunity can be adoptively transferred with T lymphocytes from Lm-immune donors. The Lm-immune cells are believed to produce macrophage-activating lymphokines, which leads to the eventual macrophage-dependent eradication of the bacterium. Increasing evidence suggests that immunity to Lm resides exclusively within the CD8+ T cell subset. It is possible that the Lm-immune CD8+ T cells function to release sequestered Lm from nonprofessional phagocytes to awaiting activated macrophage populations. This study was conducted to determine if listeriolysin O (LLO), which is an essential determinant of Lm pathogenicity, is also a target of the antilisterial immune response. We have found that target cells infected with a LLO+ Lm strain are lysed by Lm-immune cytotoxic cells, whereas target cells infected with a LLO- Lm mutant, or pulsed with a heat-killed Lm preparation, are not lysed by the Lm-immune effector cells. We have used a Bacillus subtilis (Bs) construct that expresses the LLO gene product and found that target cells infected with the LLO+ Bs construct are lysed by antilisterial cytotoxic cells. The antilisterial cytotoxic response is targeted against LLO, in that we have also used a Bs construct that expresses the perfringolysin (PLO) gene product and found that target cells infected with the PLO+ Bs are not lysed by antilisterial cytotoxic effector cells. These data strongly suggest that LLO is a target antigen of antilisterial immunity and may represent the dominant target during the expression of the immune response to Lm.