We have recently reported that NY-ESO-1-specific naive CD4+ T cell precursors exist in most individuals but are suppressed by CD4+CD25+ regulatory T cells (Tregs), while memory CD4+ T cell effectors against NY-ESO-1 are found only in cancer patients with spontaneous Ab responses to NY-ESO-1. In this study, we have analyzed mechanisms of CD4+ T cell induction following peptide vaccination in relation to susceptibility to Tregs. Specific HLA-DP4-restricted CD4+ T cell responses were elicited after vaccination with NY-ESO-1(157-170) peptide (emulsified in IFA) in patients with NY-ESO-1-expressing epithelial ovarian cancer. These vaccine-induced CD4+ T cells were detectable from effector/memory populations without requirement for in vitro CD4+CD25+ T cell depletion. However, they were only able to recognize NY-ESO-1(157-170) peptide but not naturally processed NY-ESO-1 protein and had much lower avidity compared with NY-ESO-1-specific pre-existing naive CD4+CD25- T cell precursors or spontaneously induced CD4+ T cell effectors of cancer patients with NY-ESO-1 Ab. We propose that vaccination with NY-ESO-1(157-170) peptide recruits low-avidity T cells with low sensitivity to Tregs and fails to modulate the suppressive effect of Tregs on high-avidity NY-ESO-1-specific T cell precursors.