Squamous cell carcinomas of the oropharynx (SCCO) are often infected with oncogenic human papilloma virus (HPV) subtype 16. To determine the frequency of T cells specific for human leukocyte antigen (HLA)-A2.1 restricted HPV16 E7 protein-derived epitopes, tetramer analysis was performed using peripheral blood lymphocytes of 20 HLA-A2.1+ patients and 20 HLA-A2.1+ healthy individuals. Tetramers specific for 3 HPV16 peptides (E711–20, E782–90 and E786–93), an influenza matrix peptide (a model recall antigen) or an HIV reverse transcriptase peptide (a model novel antigen) were used in multicolor flow analysis. The HPV-specific T-cell frequencies were correlated with the HPV16 E7 and p16 status in tumor sections. In vitro stimulation (IVS) with autologous dendritic cells (DC) pulsed with HPV16 E7 epitopes was performed to demonstrate proliferation and antitumor activity of the HPV-responsive T cells. Frequencies of CD8+ T cells specific for HPV16 E7 peptides were not significantly different in patients with SCCO relative to normal donors. However, patients with tumors expressing HPV16 E7 (60%) and p16 (50%) had an increased frequency (p < 0.05) of T cells specific for the E711–20 epitope compared to those with tumors negative for both markers. HPV16 E711–20 and HPV16 E786–93 specific T cells were expandable upon IVS with cognate peptide-pulsed DC and were reactive against peptide-pulsed targets or, in case of the E711–20 epitope-specific T cells, against HPV16 E7 expressing CaSki cell line. Thus, in patients with HPV16+ SCCO, precursor T cells specific for E711–20 epitope are present (1/3,947) in the circulation, are responsive to stimulation with the cognate viral peptide and recognize in vitro HPV16 E7+ tumor cells. Further studies have to elucidate why those T cells are unable to eliminate the tumor in vivo and this might also allow for finding potential strategies that will increase the chances of developing a future HPV-based vaccine in patients with SCCO. © 2005 Wiley-Liss, Inc.