Endogenously synthesized polyepitope protein targeted to the endocytic/lysosomal compartment facilitates presentation of multiple epitopes to CD4+ T cells. Autologous LCL cells infected with different vaccinia virus recombinants (Vacc.POLY, Vacc.ER-POLY, Vacc.INV-POLY, Vacc.ER-POLY-LAMP, Vacc.EBNA2, Vacc.BHRF1, Vacc.EBNA1, and Vacc.TK−) were used as targets in standard CTL assays. CTL clones used in these assays were LC27 (EBNA2-specific), SBAg1 (BHRF1-specific), and DM2 (EBNA1-specific). LC/Ag876, SB29f, and DM/B95.8 LCLs were used as host cells for vaccinia virus constructs for LC27, SBAg1, and DM2 CTL clones, respectively. The LC/Ag876 LCL is not recognized by LC27, since this cell line is infected with a type 2 EBV which expresses a variant EBNA2 epitope sequence. SB29f LCL cells are negative for BHRF1 antigen, while the EBNA1 epitope is not endogenously processed by DM/B95.8 LCL cells, and thus these cells are not recognized by SBAg1 and DM2 CTL clones, respectively.