EBV-infected cells and EBV-associated tumors may evade CTL recognition by defective antigen processing, resulting in poor presentation of CTL epitopes. Since the proteasome is the major source of MHC class I–presented peptides, we analyzed the effect of proteasome inhibitors on the expression of surface HLA class I and the generation of EBV-derived CTL epitopes presented by the HLA-A2 and HLA-A11 alleles. Treatment with covalent and reversible inhibitors of the proteasome partially reduced the total and allele-specific expression of surface HLA class I in EBV-carrying LCLs. HLA-A2 expression was also decreased by treatment with leupeptin and bestatin, while HLA-A11 expression was affected by treatment with phenanthroline. Despite their general inhibitory effect on HLA class I expression, all proteasome inhibitors tested enhanced the presentation of 2 subdominant HLA-A2 epitopes from EBV LMP1 and LMP2, while the presentation of the immunodominant HLA-A11-restricted epitope from EBNA4 was inhibited by MG132 and lactacystin and increased by ZL3VS. Treatment with ZL3VS restored the presentation of endogenously expressed EBNA4 in 1 HLA-A11-positive BL cell line. These findings suggest that specific inhibitors of the proteasome may be used to increase the antigenicity of virus-infected and malignant cells that are per se inefficient at generating particular CTL target epitopes. © 2002 Wiley-Liss, Inc.