Human papilloma virus (HPV) type 16 is found in the majority of cervical cancer patients and the transforming protein E7 is consistently expressed in cancer cells, making it a potential target for immune attack. In this study we have investigated whether E7 gains access to the MHC class I processing pathway and provides cytotoxic T lymphocyte (CTL) stimulating peptide epitopes. CTL were induced in H-2b mice by immunization with recombinant vaccinia virus expressing E7 (Vac-E7). To map CTL recognition, natural peptides were purified from cells expressing either intact or truncated E7 protein. Following peptide separation by HPLC one major CTL epitope was detected and truncated constructs localized this epitope to the C-terminal region. Mapping with synthetic peptides indicated that residues 49-57 (RAHYNIVTF) were recognised by anti-E7 CTL. Synthetic 49-57 peptide was used to induce CTL, which recognized the same HPLC purified natural peptide fractions as anti-E7 CTL. Binding motifs for H-2b class I molecules did not predict residues 49-57 to be a CTL epitope, but instead the sequence 21-28 (DLYCYEQL) which contains a Kb anchor motif. Synthetic 21-28 peptide was found to bind to Kb class I molecules and readily induced CTL, indicating that the T cell repertoire of H-2b mice can recognize this epitope. However, these CTL did not recognize peptides isolated from E7 expressing cells, showing that natural processing did not produce detectable levels of the 21-28 epitope. Together, the data demonstrate that an unexpected E7 peptide can function as a major CTL epitope.