Listeria monocytogenes is a pathogenic intracellular bacterium that secretes proteins into the cytosol of host cells. A major secreted protein, p60, is processed by the host cell into the nonamer peptides p60 217-225 and p60 449-457, which are presented to CTL by H-2Kd MHC class I molecules. Herein, we use two membrane permeable peptide aldehyde protease inhibitors, LLnL and Z-LLF, to inhibit cytosolic proteolysis in L. monocytogenes-infected cells. These inhibitors, which have been shown to inhibit proteasomes, completely abrogate cytosolic p60 degradation. The effect of LLnL and Z-LLF on p60 epitope generation was determined by acid-eluting, HPLC-purifying, and quantifying p60 217-225 and p60 449-457 from infected cells. We show a direct linkage between p60 degradation and epitope generation. However, the two inhibitors have quantitatively different effects on the generation of the two epitopes. Our findings implicate proteasomes in the earliest stages of Ag degradation and suggest that different CTL epitopes can be generated by distinct proteolytic processes.