Immunotherapy for cancer relies on the identification of tumor antigens and efficacy of antitumor immune responses. Serological analysis of recombinant cDNA libraries (SEREX), which is based on the spontaneous humoral responses against potential tumor antigens, has provided a novel strategy for searching novel tumor-associated candidates. Through SEREX analysis, we have identified 24 distinct gene clones by immunoscreening of a cDNA library derived from an ovarian cancer patient. Among these genes, a novel gene, OVA66, was found to be expressed significantly higher in carcinoma samples from cancer patients than in normal controls. Comparing humoral responses to OVA66 between tumor patients and healthy donors, it has been shown that the IgG level against OVA66 was significantly elevated in the serum of cancer patients from different histological types of cancer. To determine whether SEREX-defined OVA66 can trigger promising cytotoxic T lymphocyte (CTL) responses, human leukocyte antigen (HLA)-A*0201-restricted T-cell epitopes were predicted through a computational algorithm. Of four predicted peptides, p306-314 (L235) possesses the ability to induce efficient peripheral blood lymphocyte (PBL)-derived CTL responses capable of specifically recognizing peptide-pulsed T2 cells and lysing carcinoma cell lines expressing both HLA-A2 and OVA66 as determined by cytotoxicity and enzyme-linked immunospot assay (ELISPOT). Taken together, our results demonstrate that the SEREX-defined tumor-associated antigen OVA66 can elicit humoral immunity and may also serve as a potential candidate for T-cell-based immunotherapy for cancer. © 2008 Japanese Cancer Association.