Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is an attractive target for immunotherapy against EBV-associated malignancies because it is expressed in all EBV-positive cells. Although CD8+ cytotoxic T-lymphocyte (CTL) epitope presentation is largely prevented by its glycine-alanine-repeat domain (GAr), the use of mRNA-transduced dendritic cells (DCs) would offer the advantage of priming EBNA1-specific CTLs. After stimulation with GAr-containing EBNA1-transduced monocyte-derived DCs, two EBNA1-specific CTL clones, B5 and C6, were isolated successfully from a healthy donor. These CTLs recognize peptides in the context of HLA-B*3501 and HLA-Cw*0303, respectively. A novel epitope, FVYGGSKTSL, was then identified, presented by both HLA-Cw*0303 and -Cw*0304, which are expressed by >35% of Japanese, >20% of Northern Han Chinese and >25% of Caucasians. The mixed lymphocyte-peptide culture method revealed that FVYGGSKTSL-specific CTL-precursor frequencies in HLA-Cw*0303- or -Cw*0304-positive donors were between 1x10(-5) and 1x10(-4) CD8+ T cells. Moreover, both CTL clones inhibited growth of HLA-matched EBV-transformed B lymphocytes in vitro, and B5 CTLs produced a gamma interferon response to EBNA1-expressing gastric carcinoma cells in the context of HLA-Cw*0303. These data demonstrate that EBNA1 mRNA-transduced DCs may be useful tools for inducing EBNA1-specific CTLs that might be of clinical interest for CTL therapy of EBV-associated malignancies.