The purpose of this study was to identify polymorphic residues of DQ2 beta chains which are involved in the epitopes recognized by DQ2-reactive mAbs. Binding studies were performed on a panel of DQ2 mutant cells made by transfection of a DQ (alpha 1*0501, beta 1*0301)-positive B-LCL with DQB1 genes encoding either wild-type or mutated DQ beta 1 *0202. Several aa substitutions were found to influence the binding of DQ2 mAbs. All but one of the mAbs were clearly sensitive to the introduced aa substitutions, but individual mAbs were differentially influenced, suggesting that they recognized different epitopes on the DQ2 molecule. Substitutions in positions 30 and 37 of the peptide-binding cleft were also found to influence the binding of some mAbs. Second, two mAbs, NFLD.M71 and NFLD.M102, which bound to DQ(alpha 1*0501, beta 1*0202) expressed in human cells and were sensitive to the introduction of aa substitutions in the cleft, bound weakly to the DQ(alpha 1*0501, beta 1*0202) heterodimer when expressed in a transfected murine NIH 3T3 cell line. Together this indicates that some DQ2-reactive mAbs may recognize peptide-dependent epitopes.