The peptide binding specificities of HLA-DRB1* 0401, DRB1* 0101, and DRB1* 0701 have been analyzed by the use of large collections of synthetic peptides corresponding to naturally occurring sequences. The results demonstrated that nearly all peptides binding to these DR molecules bear a motif characterized by a large aromatic or hydrophobic residue in position 1 (Y, F, W, L, I, V, M) and a small, noncharged residue in position 6 (S, T, C, A, P, V, I, L, M). In addition, allele-specific secondary effects and secondary anchors were defined, and these parameters were utilized to derive allele-specific motifs and algorithms. By the combined use of such algorithms, peptides capable of degenerate DRB1* 0101, DRB1* 0401, and DRB1* 0701 binding were identified. Additional experiments utilizing a panel of quantitative assays specific for nine additional common DR molecules identified a large set of DR molecules, which includes at least the DRB1* 0101, DRB1* 0401, DRB1* 0701, DRB5* 0101, DRB1* 1501, DRB1* 0901, and DRB1* 1302 allelic products, characterized by overlapping peptide-binding repertoires. These results have implications for understanding the molecular interactions involved in peptide-DR binding, as well as the genetic and structural basis of MHC polymorphism. These results also have potential practical implications for the development of epitope-based prophylactic and therapeutic vaccines.