Whereas tumor infiltration by T effectors is generally associated with a more favorable prognosis, the accumulation of CD4⁺ regulatory T cells (Treg) within tumors is instead often associated with poor disease outcome. Because approaches to improve antitumor immunity aim, on one hand, at expanding tumor antigen-specific T cells and, on the other, at eliminating or inactivating Treg, an outstanding question is whether, and to what extent, tumor antigen-specific CD4⁺ T effectors present at tumor sites overlap with tumor-associated Treg. Here, we used MHC class II/peptide tetramers incorporating an immunodominant peptide from the human tumor-specific antigen NY-ESO-1 to assess antigen-specific CD4⁺ T cells among conventional CD4⁺ T effectors and Treg at sites of ovarian cancer. We found that, in patients who spontaneously respond to the antigen, the frequency of NY-ESO-1 tetramer⁺ cells detected ex vivo was highly enriched in tumors as compared with the periphery. At tumor sites, NY-ESO-1 tetramer⁺ cells were detected concomitantly with high proportions of Treg but were distinct from the latter and displayed characteristics of T_H1 effectors. Thus, even in the presence of high proportions of Treg, tumor antigen-specific CD4⁺ T cells can accumulate in ovarian tumors and maintain an effector phenotype. Cancer Immunol Res; 1(5); 303–8. ©2013 AACR.