Previously, we identified human papillomavirus type 16 (HPV-16) E5 as a tumor rejection antigen that can induce cytotoxic T lymphocytes (CTLs) to protect against tumor growth (D. W. Liu et al., J. Virol. 74:9083-9089, 2000). In the present study, we further mapped the CTL epitope of E5 protein by analyzing E5-specific CD8(+) gamma interferon-positive (IFN-gamma(+)) double-positive cells in C57BL/6 mice with flow cytometry. The results showed the region spanning amino acids 25 to 33 (VCLLIRPLL) contained the potential D(b)-restricted CTL epitope. Subsequently, to determine whether peptide E5 25-33-based vaccination could induce E5-specific CTL activity, syngeneic animals received E5 25-33 emulsified with either CpG oligodeoxynucleotide (CpG ODN 1826) or Freund's adjuvant, and the growth of the tumors was monitored. The results showed that although both adjuvants induced E5-specific CD8(+) IFN-gamma(+) T cells and eradicated E5-containing tumor growth, CpG ODN was found to stimulate stronger CTL response than Freund's adjuvant. We also compared the immune response of the effector/memory/recall phase induced by E5 25-33 peptide or by E5 protein that was synthesized in vivo by adenovirus-based E5 gene delivery. E5 25-33 peptide plus CpG ODN was shown to be a superior vaccine compared to the adenovirus-based E5 gene. Interestingly, their chronological patterns of immune response were similar, suggesting that E5 25-33 is a major CTL peptide of E5 protein.