The NY-ESO-1 antigen is expressed in a significant proportion of patients with epithelial ovarian cancer (EOC) and appears to be an ideal target for immunotherapy. In order to elucidate the nature of the HLA-DPB1*0401/0402 (DP4+)-restricted CD4+ immune response in patients with NY-ESO-1-expressing EOC, peripheral blood CD4+ T cells from HLA-DP4+ patients were stimulated with the NY-ESO-1 epitope 157-170 and tested for the release of type 1 (IFN-gamma) and type 2 (IL-5) cytokines in enzyme-linked immunospot assays. Of 14 DP4+ EOC patients who tested seronegative for NY-ESO-1, 3 patients had a detectable CD4+ T cell response to NY-ESO-1 epitope 157-170 by IFN-gamma ELISPOT assay. Six of 10 DP4+ EOC patients with serum antibodies to NY-ESO-1 had CD4+ T cell responses to NY-ESO-1 epitope 157-170 by IFN-gamma assay. Six patients had mixed Th1/Th2 CD4+ T cell responses to NY-ESO-1 epitope 157-170 regardless of their antibody response to NY-ESO-1. Four EOC patients had Th1 cells expressing IFN-gamma, but not IL-5. This suggests that the NY-ESO-1 epitope 157-170 stimulates both Th1 and Th2 type CD4+ T cell responses in EOC patients. These data suggest the NY-ESO-1 epitope 157-170 has a key role in the induction of cellular and humoral immune responses against NY-ESO-1-expressing EOC tumors. Our study supports the relevance of cancer vaccine trials with the NY-ESO-1 epitope 157-170 in HLA-DP4+ EOC patients with NY-ESO-1-expressing tumors and strategies to improve Th1-dominated tumor-reactive CD4+ T cell bias.