Human papillomavirus type 16 (HPV-16) is strongly associated with cervical cancer. HPV-16 cytotoxic T lymphocyte (CTL) epitopes may be good candidates for the development of an antitumor peptide vaccine. A set of 240 overlapping peptides nine amino acids in length with an eight amino acid overlap covering the entire sequence of the two viral oncogenes E6 and E7 was synthesized and tested for its ability to bind to the most common human leukocyte antigen class I molecule HLA-A2.1. Binding was measured with the human processing defective cell line T2, which expresses high numbers of empty HLA-A2.1 molecules that are unstable at 37 degrees C. These empty molecules can be stabilized by exogenously added peptides, and the extent of stabilization, measured by cell surface HLA-A2.1-specific staining, can be taken as a measure of the relative HLA-A2.1 binding affinity. Following this analysis, several HLA-A2.1 binding peptides were pinpointed. Preliminary data suggest that at least one of the high-affinity-binding peptides identified is immunogenic even in an in vitro priming protocol, underlining the feasibility of the method described here to identify the immunogenic peptides and potential candidates for CTL peptide-based vaccines.