Listeria monocytogenes (L. monocytogenes) is a pathogenic bacterium, and subclinical infection in mice is utilized as a prototypic model to investigate the development and expression of acquired resistance to facultative intracellular organisms. A key virulence factor of L. monocytogenes is the hemolysin listeriolysin O (LLO), and BALB/c mice immunized with hemolysin-secreting strains of L. monocytogenes develop specific acquired resistance, while mice immunized with hemolysin-negative strains or non-viable preparations of L. monocytogenes do not develop a protective immune response. Adoptive transfer studies show that L. monocytogenes-immune CD8+ T cells mediate acquired resistance. The L. monocytogenes-immune CD8+ population is cytotoxic, and target cells infected with hemolysin-secreting strains of L. monocytogenes are lysed, while target cells infected with hemolysin-negative strains or non-viable preparations of L. monocytogenes are not lysed. MHC class Ia and Ib molecules present L. monocytogenes-derived peptides, and we have identified Qa-Ib, a T-region-encoded MHC class Ib molecule, as a restriction element for L. monocytogenes-specific CD8+ CTL. MHC class Ib-restricted CTL are stimulated following infection with L. monocytogenes and are a significant component of the total MHC class I-restricted CTL population. These findings support the observation that cytoplasmic L. monocytogenes-derived antigens are endogenously processed and presented in association with MHC class Ia and Ib molecules to CD8+ effector cells, and that both populations of effector cells contribute to the immune response to this intracellular pathogen.