A proportion of cancer patients naturally develop CD4⁺ T-helper type 1 (Th1) cell responses to NY-ESO-1 that correlate with anti–NY-ESO-1 serum antibodies. To address the role of T-cell regulation in the control of spontaneous tumor immunity, we analyzed NY-ESO-1–specific Th1 cell induction before or after depletion of CD4⁺CD25⁺ T cells in vitro. While Th1 cells were generated in the presence of CD25⁺ T cells in cancer patients seropositive for NY-ESO-1, seronegative cancer patients and healthy donors required CD25⁺ T-cell depletion for in vitro induction of NY-ESO-1–specific Th1 cells. In vitro, newly generated NY-ESO-1–specific Th1 cells were derived from naive precursors, whereas preexisting memory populations were detectable exclusively in patients with NY-ESO-1 antibody. Memory populations were less sensitive than naive populations to CD4⁺CD25⁺ regulatory T cells. We propose that CD4⁺CD25⁺ regulatory T cells are involved in the generation and regulation of NY-ESO-1–specific antitumor immunity.