Graves' disease (GD) is an autoimmune disease of the thyroid gland. Genes of, or closely associated to, the HLA complex are assumed to contribute to the genetic predisposition to GD. We have previously reported an increased frequency of HLA-DR3/DQ2 in Caucasian patients with GD, and recently the importance of Dw24 encoded by DRB3 gene has been suggested. To further investigate the associations of GD and these genes, 94 unrelated patients with GD and 75 control subjects were typed for HLA-DRB3, -DRB1, -DQA1, and -DQB1, using sequence-specific oligonucleotide probes to analyze polymerase chain reaction amplified DNA (PCR-SSO). Three findings emerged from these studies. 1) The frequency of subjects positive for DQA1*0501 (GD, 73.4% vs. control 42.7%, P = 0.0001, Pc < 0.001, RR = 3.71) was significantly increased among patients. The frequency of DR3 (GD, 34.0% vs. control 17.3%, P = 0.0146, RR = 2.46), which is in tight linkage disequilibrium with DQA1*0501, was also increased; however, it was not significant when the P value was corrected for the number of antigens tested. Neither DQB1 nor DRB3 alleles were significantly increased in frequency. 2) After exclusion of DR3-positive subjects, DQA1*0501 was still significantly increased (GD, 59.7% vs. control 30.6%, P = 0.0012, Pc < 0.01, RR = 3.35) among patients. 3) The distributions of Dw24 and Dw25,26 (Dw25 or Dw26) did not differ between patients and controls on either DR3 positive or negative groups. These findings suggest that DQA1*0501, or a closely associated unknown gene, confers susceptibility to GD, while Dw24 is not directly involved. The importance of DR3, however, remains to be elucidated, because of the fixed linkage with DQA1*0501.