Assays to measure the binding capacity of peptides for HLA-DQA1* 0501/B* 0201 (DQ2. 3) and DQA1* 0301/B* 0302 (DQ3. 2) were developed using solubilized MHC molecules purified from EBV-transformed cell lines. These quantitative assays, based on the principle of the inhibition of binding of a high-affinity radiolabeled ligand, were validated by examining the binding capacity of known DQ-restricted epitopes or ligands. The availability of these assays allowed an investigation of patterns of cross-reactivity between different DQ molecules and with various common DR molecules. DQ2. 3 and DQ3. 2 were found to have significantly overlapping peptide binding repertoires. Specifically, of 13 peptides that bound either DQ2. 3 or DQ3. 2, nine (69.2%) bound both. The molecular basis of this high degree of cross-reactivity was further investigated with panels of single substitution analogs of the thyroid peroxidase 632–645Y epitope. It was found that DQ2. 3 and DQ3. 2 bind the same ligands by using similar anchor residues but different registers. These data suggest that in analogy to what was previously described for HLA-DR molecules, HLA-DQ supertypes characterized by largely overlapping binding repertoires can be defined. In light of the known linkage of both HLA-DQ2. 3 and-DQ3. 2 with insulin-dependent diabetes mellitus and celiac disease, these results might have important implications for understanding HLA class II autoimmune disease associations.