Graves' disease (GD) is associated with HLA-DR3 (DRB1*03) in Caucasians, but the exact amino-acid sequence in the DR beta1 chain conferring susceptibility to GD is unknown. Therefore, the aim of our study was to identify the critical sequence among the HLA-DRB1 amino-acid residues occupying the peptide-binding pocket, which conferred susceptibility to GD. We sequenced the HLA-DRB1 locus in 208 Caucasian GD patients and 149 Caucasian controls. Sequence analysis showed an increased frequency of DR beta-Arg-74 in GD patients compared to controls (41.8 and 13.4%, respectively; P=2.3 x 10(-8), OR=4.6). Moreover, subset analyses showed that DR beta-Arg-74 was also significantly more frequent in the HLA-DR3 negative GD patients than in controls (7.6 vs 0.8%, P=0.02, OR=10.5), suggesting that the association with DR beta-Arg-74 is independent of the association with HLA-DR3. Structural modeling studies demonstrated that the change at position 74 from the neutral amino acids Ala or Gln to the positively charged amino-acid Arg significantly modifies the three-dimensional structure of the DR peptide-binding pocket. Our results suggested that structural heterogeneity of the DR beta-chain peptide-binding pocket P4 at residue 74 predispose some at risk individuals to GD.