Nasopharyngeal carcinoma (NPC) is a human epithelial tumor with a high incidence in Southern Chinese population, with contributions from Epstein-Barr virus (EBV), human leukocyte antigen (HLA), and environmental factors to its etiology. It has been shown previously that the recognition of immediate-early transactivator Rta of EBV by CD8+ T cells may have a significant impact on controlling EBV and, indirectly, NPC. The current study used two computer-aided prediction methods and competition-based HLA-peptide binding assays to screen for HLA B2704/B4601/B5801 restricted T-cell epitopes derived from Rta. HLA tetrameric complexes containing these potential T-cell epitopes were synthesized. Rta-specific CD8+ T-cell responses in healthy virus carriers were then defined by these tetramers and IFN-gamma ELISPOT assays. We clearly demonstrated that healthy virus carriers have detectable Rta-specific CD8+ T cells restricted by B2704 in the circulation. However, there were no B4601/B5801 tetramer-reactive T cells specific for Rta in the peripheral blood of matched/mismatched donors. On the other hand, B4601 tetramers containing the computer-predicted B4601 binder EBNA3A (318-326) showed detectable tetramer-reactive T cells in the circulation of healthy virus carriers. topes also elicited IFN-gamma responses as detected by ELISPOT.