Immunodominance in CD8+ T cell responses against Listeria monocytogenes is a well-recognized but still not fully understood phenomenon. From listeriolysin, the major virulence factor of L. monocytogenes, only a single epitope, pLLO91-99, is presented by MHC class I molecules in BALB/c mice which dominates the cytotoxic T cell response against this bacterial pathogen. To obtain more insights into the molecular and cellular mechanisms underlying immunodominance of this particular epitope, we compared the various steps involved in the presentation and recognition of pLLO91-99 derived from a wild-type toxin with an equivalent epitope from a mutated toxin. This fully functional variant contains within the pLLO91-99 epitope a conservative isoleucine to alanine replacement at the C-terminal anchor residue which results in loss of antigenicity. The binding properties of the variant peptide to soluble Kd remained unaffected and cytotoxic T cells capable of recognizing the pLLO99A/Kd complex were detectable in BALB/c mice. However, such T cells required higher concentrations of antigen in order to be optimally activated in vitro. A comparison between the TAP translocation efficiency of wild-type and mutant peptide demonstrated that the mutation at the C-terminus leads to a reduced transportation rate. Furthermore, the amino acid substitution changes the in vitro proteasomal cleavage pattern, resulting in a reduced liberation of the correct peptide from a polypeptide precursor. Thus, in all assays employed the immunodominant epitope performs optimally while the variant was found to be inferior. The synergy of all these steps most likely is the decisive factor in the immunodominance of pLLO91-99.