NY-ESO-1 and LAGE-1 represent highly homologous cancer-germline Ags frequently coexpressed by many human cancers, but not by normal tissues, except testis. In contrast to NY-ESO-1, little is known about spontaneous immune responses to LAGE-1. In the current study, we report on spontaneous LAGE-1-specific CD4(+) T cells isolated from PBLs of patients with advanced LAGE-1(+)/NY-ESO-1(+) melanoma and directed against three promiscuous and immunodominant epitopes. Strikingly, although the three LAGE-1-derived epitopes are highly homologous to NY-ESO-1-derived epitopes, LAGE-1-specific CD4(+) T cells did not cross-react with NY-ESO-1. LAGE-1-specific CD4(+) T cells produced Th1-type and/or Th2-type cytokines and did not exert inhibitory effects on allogenic T cells. We observed that most patients with spontaneous NY-ESO-1-specific responses exhibited spontaneous CD4(+) T cell responses to at least one of the three immunodominant LAGE-1 epitopes. Additionally, nearly half of the patients with spontaneous LAGE-1-specific CD4(+) T cell responses had circulating LAGE-1-specific Abs that recognized epitopes located in the C-terminal portion of LAGE-1, which is distinct from NY-ESO-1. Collectively, our findings define the hierarchy of immunodominance of spontaneous LAGE-1-specific CD4(+) T cell responses in patients with advanced melanoma. These findings demonstrate the capability of LAGE-1 to stimulate integrated cellular and humoral immune responses that do not cross-react with NY-ESO-1. Therefore, they provide a strong rationale for the inclusion of LAGE-1 peptides or protein in vaccine trials for patients with NY-ESO-1(+)/LAGE-1(+) tumors.