A large number of human tumor antigens recognized by CD8+ cytotoxic T lymphocytes (CTL) have been identified. Some of them have been employed in clinical trials and have achieved some objective responses. However, little is known about those that are recognized by CD4+ T cells, except for a very few that were identified from melanomas. Previously, we reported that an oral squamous cell carcinoma (SCC) cell line, OSC-20, was effectively lysed by HLA-DRB1*08032 (HLA-DR8)-restricted autologous CD4+ T cell line, TcOSC-20. In this study, we performed two steps of chromatographic purification of the tumor cell lysate in combination with mass spectrometry. We found one reverse-phase high-performance liquid chromatography (RP-HPLC) fraction that was effectively recognized by the T cells. We analyzed the fraction by nano-liquid chromatography/electrospray ionization ion trap mass spectrometry (LC/MS/MS) and found six representative ions. We could determine the primary amino acid sequence of each of the six ions. Three of them contained a potential HLA-DR8 binding motif, and TcOSC-20 showed a rather strong cytotoxic response to one of the synthetic peptides, namely, amino acid residues 321-336 of human alpha-enolase. Thus, several gene products of squamous cancer cells are endogenously processed and may be presented on HLA class II molecules, so that they could constitute target molecules for autologous CD4+ T cells.