The cancer testis (CT) antigen HCA587 is highly expressed in human hepatocellular carcinoma (HCC) and induces specific T-cell responses in a significant proportion of HCC patients. To explore its potential in cancer immunotherapy, a reverse immunology approach was adopted to identify HCA587-derived HLA-A*0201-restricted epitopes. Multiple peptides with a top ranking in various prediction programs were thus synthesized and three of them-p248-256, p140-149 and p144-152-were found to bind to HLA-A*0201 molecules with a high affinity and effectively induced a recall response of CD8(+) T cells, which were either primed in vitro with the HCA587 antigen or directly isolated from HCC patients bearing HCA587(+) tumors. Notably, these peptide-specific CD8(+) T cells exhibited potent cytotoxic activity over A587(+) tumor cells. Taken together, the present study has identified three new HLA-A*0201-restricted cytotoxic T cell epitopes in the CT antigen HCA587, which may serve as targets for peptide-based immunotherapy for HCC patients. (C) 2008 Elsevier Inc. All rights reserved.