Listeria monocytogenes is a Gram-positive bacterium which grows in the cytoplasm of eukaryotic cells and can cause severe disease in immunocompromised individuals^1,2. In murine systems CD8⁺T lymphocytes have been shown to be important effectors of acquired protective immunity against L. monocytogenes^3–5. Class I MHC-restricted CD8⁺ cytotoxic T lymphocytes (CTL), which lyse J774 macrophage-like targets infected with L. monocytogenes, are induced following in vivo injection of live organisms. Natural peptide epitopes derived from L. monocytogenes can be acid-extracted from heavily infected BALB/c spleens and detected by CTL. A CTL clone, B9, derived from a (BALB/c x C57BL/6)F₁ (H–2^dxb) mouse, recognizes one of these natural epitopes in an H–2K^d-restricted fashion. B9 also recognizes P815 (H–2^d) mas-tocytoma cells transfected with the listeriolysin gene. To identify the region of the listeriolysin recognized by CTL we used the H–2K^d peptide-binding motif described by Rammensee and colleagues⁶ to synthesize 11 nonamer peptides. One of these peptides, listeriolysin 91–99, was recognized very efficiently by B9. This represents the first identified class I MHC-restricted epitope of bacteria and demonstrates the utility of the allele-specific motif for predicting CTL epitopes.