A major H2-Kd-presented epitope for antilisterial cytotoxic T lymphocytes (CTLs) is the nanomer peptide which corresponds to the amino acid 91 to 99 (aa91-99) sequence from listeriolysin O (LLO). Although the LLO sequence contains at least five additional nanomer peptides which also satisfy the H2-Kd binding motif, aa91-99 is the only LLO-derived target peptide that is recognized by antilisterial CTLs following infection of BALB/c mice with Listeria monocytogenes. In order to investigate further the immunodominance of the LLO aa91-99 epitope following endogenous processing of LLO, we introduced a point mutation in hly (the gene for LLO) which results in a conservative Y-to-F substitution for the anchor residue at position 2 within the aa91-99 sequence. This "92F" L. monocytogenes mutant produces biologically active LLO and is phenotypically indistinct from wild-type L. monocytogenes in terms of intracellular growth in vitro and virulence in vivo. BALB/c mice actively immunized with the 92F L. monocytogenes mutant are protected against challenge with wild-type L. monocytogenes. Antilisterial CTLs from mice immunized with the 92F mutant lyse targets infected with L. monocytogenes; however, these CTLs do not lyse target cells pulsed with either the LLO aa91-99 peptide, other LLO-derived peptides which satisfy the H2-Kd binding motif, or a peptide corresponding to the LLO aa91-92F-99 sequence. Target cells pulsed with the LLO aa91-92F-99 peptide are, however, lysed by wild-type LLO aa91-99-specific cytotoxic cells. Thus, a conservative amino acid change in the first anchor residue of the immunodominant aa91-99 sequence of LLO eliminates the induction of the cytotoxic cell response to this epitope as well as to any of the other candidate LLO-derived peptides which fit the H2-Kd binding motif. The lack of anti-LLO-specific CTLs following immunization with the 92F mutant does not appear, however, to influence the protective antilisterial immune response.