The NY-ESO-1 gene product is expressed by a range of human tumors and is recognized by antibodies from sera of cancer patients with NY-ESO-1-expressing tumors. The NY-ESO-1 gene also encodes several MHC class I- and MHC class II-restricted tumor epitopes recognized by T lymphocytes. In particular, we previously reported that the NY-ESO-1 119–143 peptide contains at least two HLA-DRB1*0401-presented epitopes that are recognized by melanoma-reactive CD4+ T cells. Here we report that the NY-ESO-1 119–143 peptide can be presented in the context of multiple HLA-DR alleles to stimulate tumor-reactive CD4+ T cells. The NY-ESO-1 119–143 peptide is able to bind to several DR molecules. The NY-ESO-1 119–143 peptide is also capable of inducing specific CD4+ T cells in vitro from peripheral blood lymphocytes of normal donors and patients with melanoma who express these HLA-DR alleles. These CD4+ T cells recognize NY-ESO-1⁺, HLA-matched or autologous melanoma cell lines, as well as autologous antigen-presenting cells fed with the NY-ESO-1 protein. We also demonstrate that the NY-ESO-1 119–143 peptide stimulates in vitro both Th1-type and Th2-type CD4+ T-cell responses from peripheral blood lymphocytes of normal donors and melanoma patients. Taken together, these data suggest a key role of the NY-ESO-1 119–143 peptide sequence in the induction of cellular and humoral responses against NY-ESO-1-expressing tumors. They support the relevance of cancer vaccine trials with the NY-ESO-1 119–143 peptide in the large number of cancer patients with NY-ESO-1-expressing tumors.