To understand the rules determining peptide binding to the celiac disease and type 1 diabetes mellitus associated HLA-DQ2 molecule, we have studied in detail the binding of a peptide OVA 258–276Y (IINFEKLTEWTSSNVMEERY) which exhibitsstrong binding to DQ2. First we tested a set of N- and C-terminal truncated variants, and found the core binding region to comprise residues 267–276Y. Single alanine substitution analysis of the OVA 267–276Y peptide revealed thatreplacements of V₂₇₂, E₂₇₅ and the C-terminal Y had negative effects whereas the substitution of N₂₇₁ had a positive effect. A polyalanine analogue of the OVA 267–276Y peptide with V₂₇₂, E₂₇₅ and a C-terminal Y bound at least as well as the original peptide. A variant peptide with a deletion of R₂₇₆ displayed decreased binding, suggesting that the anchor residues were out of frame in this analogue. To further characterize the residues playing a role in the binding of the OVA 267–276Y peptide to DQ2 we tested the binding ofseveral analogues with substitutions for V₂₇₂, E₂₇₅ and the C-terminal Y residue. Our results indicate that peptides binding to DQ2 have anchor residues in relative positions 4, 7 and 9 (P4, P7 and P9). Residues with negatively charged or hydrophobic aliphatic but not positively charged side chains are preferred in P4 and P7, whereas residues with bulky hydrophobic side chains are preferred in P9.