Background. Thymidylate synthase (TS), a key enzyme in DNA synthesis, is often overexpressed in cancer cells. Some chemotherapeutic agents, such as 5-fluorouracil (5-FU), act by inhibiting TS expression. We evaluated whether a novel 28-amino acid multiepitope peptide, TS/PP, that contains the sequences of three TS-derived epitopes with binding motifs for HLA-A(*)02.01 could induce a TS-directed cytotoxic T-lymphocyte (CTL) response with antitumor activity. Methods: TS/PP peptide immunologic activity in CTL lines derived from human leukocyte antigen (HLA)-A(*)02.01(+) peripheral blood mononuclear cells (PBMCs) was tested in the presence of interleukin-2 and autologous TS/PP peptide-loaded dendritic cells. Immunologic and antitumor activities of TS/PP and its toxicity were also evaluated in vivo in HLA-A(*)02.01 transgenic (HHD) mice that were vaccinated with TS/PP, control, or TS-peptide cocktail and treated with or without 5-FU chemotherapy. The mice were also inoculated subcutaneously with TS-expressing EL-4/HHD lymphoma cells to assess immune response against these tumor cells. Results: TS/PP-specific CTL lines showed a TS-multiepitopic specificity and were able to kill TS+/HLA-A(*)02.01(+) breast and colon carcinoma cells. The killing ability against target cells previously exposed to sublethal doses of 5-FU was statistically significantly greater than against untreated target cells (43.5% versus 26.5% at 25/1 effector to target ratio [Difference {diff} = 17.0]; 95% confidence interval [CI] = 12.6 to 20.4) for MDA-MB-231 breast carcinoma cells and 73.5 versus 48.5 (diff = 25.0; 95% CI = 16.2 to 33.8) for the SW-1463 colon carcinoma cells. HHD mice vaccinated with TS/PP manifested a TS-peptide-specific CTL response with no sign of autoimmunity or toxicity. Furthermore, treatment of these mice with 5-FU delayed or prevented the occurrence of tumors formed by inoculation with autologous (TS')EL-4/HHD lymphoma cells. Conclusions: The multiepitopic TS/PP vaccine induces a tumor-specific immune response in mice and is especially potent when used in combination with 5-FU-based chemotherapy.