Abstract CD8 T cells resolve intracellular pathogens by responding to pathogen-derived peptides that are presented on the cell surface by MHC class I molecules. Although most pathogens encode a large variety of antigenic peptides, protective CD8 T cell responses target usually only a few of these. To determine the mechanism by which the IFN-γ-inducible proteasome (immuno) subunits enhance the ability of specific pathogen-derived peptides to elicit CD8 T cell responses, we generated a recombinant Listeria monocytogenes strain (rLM-E1) that secretes a model Ag encompassing the immunoproteasome-dependent E1B192–200 and immunoproteasome-independent E1A234–243 epitope. Analyses of Ag presentation showed that infected gene-deficient professional APCs, lacking the immunosubunits LMP7/iβ5 and MECL-1/iβ2, processed and presented the rLM-E1-derived E1B192–200 epitope but with delayed kinetics. E1A epitope processing proceeded normally in these cells. Accordingly, infected gene-deficient mice failed to respond to the otherwise immunodominant E1B192–200 epitope but mounted normal CD8 T cell responses to E1A234–243 which was processed by the same professional APCs, from the same rLM-E1 Ag. The inability of gene-deficient mice to respond to E1B192–200 was not explained by insufficient quantities of antigenic peptide, as splenic APC of 36-h-infected gene-deficient mice that presented the two E1 epitopes at steady state levels elicited responses to both E1B192–200 and E1A234–243 when transferred into LMP7+MECL-1-deficient mice. Taken together, our findings indicate that not absolute epitope quantities but early Ag-processing kinetics determine the ability of pathogen-derived peptides to elicit CD8 T cell responses, which is of importance for rational T cell vaccine design.