Abstract Among HLA-DP specificities, HLA-DP4 specificity involves at least two molecules, HLA-DPA1*0103/DPB1*0401 (DP401) and HLA-DPA1*0103/DPB1*0402 (DP402), which differ from each other by only three residues. Together, they are present worldwide at an allelic frequency of 20–60% and are the most abundant human HLA II alleles. Strikingly, the peptide-binding specificities of these molecules have never been investigated. Hence, in this study, we report the peptide-binding motifs of both molecules. We first set up a binding assay specific for the immunopurified HLA-DP4 molecules. Using multiple sets of synthetic peptides, we successfully defined the amino acid preferences of the anchor residues. With these assays, we were also able to identify new peptide ligands from allergens and viral and tumor Ags. DP401 and DP402 exhibit very similar patterns of recognition in agreement with molecular modeling of the complexes. Pockets P1 and P6 accommodate the main anchor residues and interestingly contain only two polymorphic residues, β86 and β11, respectively. Both positions are almost dimorphic and thus produce a limited number of pocket combinations. Taken together, our results support the existence of three main binding supertypes among HLA-DP molecules and should significantly contribute to the identification of universal epitopes to be used in peptide-based vaccines for cancer, as well as for allergic or infectious diseases.