Peptide vaccination for cancer immunotherapy requires identification of peptide epitopes derived from antigenic proteins associated with tumors. Heparanase (Hpa) is broadly expressed in various advanced tumors and seems to be an attractive new tumor-associated antigen. The present study was designed to predict and identify HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitopes in the protein of human Hpa. For this purpose, HLA-A2-restricted CTL epitopes were identified using the following four-step procedure: 1) a computer-based epitope prediction from the amino acid sequence of human Hpa, 2) a peptide-binding assay to determine the affinity of the predicted protein with the HLA-A2 molecule, 3) stimulation of the primary T-cell response against the predicted peptides in vitro, and 4) testing of the induced CTLs toward different kinds of carcinoma cells expressing Hpa antigens and/or HLA-A2. The results demonstrated that, of the tested peptides, effectors induced by peptides of human Hpa containing residues 525-533 (PAFSYSFFV, Hpa525), 277-285 (KMLKSFLKA, Hpa277), and 405-413 (WLSLLFKKL, Hpa405) could effectively lyse various tumor cell lines that were Hpa-positive and HLA-A2-matched. We also found that these peptide-specific CTLs could not lyse autologous lymphocytes with low Hpa activity. Further study revealed that Hpa525, Hpa277, and Hpa405 peptides increased the frequency of IFN-gamma-producing T cells compared to a negative peptide. Our results suggest that Hpa525, Hpa277, and Hpa405 peptides are new HLA-A2-restricted CTL epitopes capable of inducing Hpa-specific CTLs in vitro. Because Hpa is expressed in most advanced malignant tumors, Hpa525, Hpa277, and Hpa405 peptide-based vaccines may be useful for the immunotherapy for patients with advanced tumors.