Previously, safety and immunogenicity of human papillomavirus type 16 (HPV16) or 18 E7-pulsed dendritic cells (DC) vaccinations were demonstrated in a dose-escalation Phase I clinical trial which enrolled ten patients diagnosed with stage IB or IIA cervical cancer (nine HPV 16-positive, one HPV 18-positive). The goal of the study was to define the T-cell epitopes of HPV 16 or 18 E7 protein in these patients in order to develop new strategies for treating HPV-associated malignancies. This was accomplished through establishing T-cell lines by stimulating peripheral blood mononuclear cells with autologous mature DC pulsed with the HPV 16 or 18 E7 protein, examining the T-cell responses using ELISPOT assays, and isolating E7-specific T-cell clones based on IFN-γ secretion. Then, the epitope was characterized in terms of its core sequence and the restriction element. Twelve T-cell lines from eight subjects (seven HPV 16-positive, one HPV 18-positive) were evaluated. Positive T-cell responses were demonstrated in four subjects (all HPV 16-positive). All four were positive for the HPV 16 E7 46-70 (EPDRAHYNIVTFCCKCDSTLRLCVQ) region. T-cell clones specific for the E7 47–70 region were isolated from one of the subjects. Further analyses revealed a novel, naturally processed, CD4 T-cell epitope, E7 58–68 (CCKCDSTLRLC), restricted by the HLA-DR17 molecule.