The structural requirements for the interaction between antigens and class I molecules was investigated through the use of a quantitative assay to measure peptide binding to different MHC class I alleles. We determined the permissiveness of the main anchors reported by Rammensee and his group for peptide binding and defined an extended motif for peptides binding to the HLA-A2.1 allele, including the role of non-anchor positions. It was found that the main anchors were necessary, but not sufficient, for good binding. Certain non-anchor positions contributed significantly to overall binding and were referred to a secondary anchors. This finding allowed a better prediction of high affinity binding peptides selected from libraries of different viral and tumor proteins. Furthermore, our data allowed correlation of the structural requirements for binding of peptides with crystallographic data of the MHC molecule. In order to characterize allele-specific motifs for a larger number of alleles, the HLA-A alleles A1, A3, A11, and A24, which represent some of the most common alleles found in different ethnic populations, were chosen. Here, most motifs were found to be highly exclusive; however, HLA-A3 and A11 shared a common motif. The defined motifs were validated further by using naturally processed peptides. Those peptides were also synthesized and tested for binding to the appropriate HLA alleles, giving a binding affinity from 0.3 to 200 nM for sequences of naturally processed peptides. Finally, a set of all possible 9-mer peptides from HPV 16 proteins were synthesized and tested for binding to the five class I alleles. For each allele, high affinity binders were identified, thus allowing for selection of possible peptide candidates for a CTL based vaccine.