Graves' disease is an autoimmune disease believed to be caused by a combination of environmental and genetic factors. One of the candidate genes is CTLA-4, a negative regulator of T cell activation. Three polymorphisms of the gene have been described, in the promoter at position -318, at position 49 in exon 1, and an (AT)n repeat within the 3'-untranslated region of exon 4. Many studies describe the association between a polymorphism of the CTLA-4 gene and autoimmune disease. To investigate the association of these CTLA-4 gene polymorphisms with each other, we analyzed the combined frequencies of each polymorphism and calculated the disequilibrium coefficients. We studied DNA samples from 120 Graves' disease (GD) patients and 80 healthy donors (NC). The exon 1 position 49 A/G polymorphism and promoter polymorphism at position -318, were typed using a PCR-restriction fragment length polymorphism method (PCR-RFLP). The polymorphic (AT)n repeat in exon 4 was determined by PCR amplification of genomic DNA, resolution of the amplified products on sequencing gels, and detection by autoradiography. There was a significant difference between GD and NC patients and occurrence of the polymorphism in exon 1 and exon 3, but not for the polymorphism in the promoter region. Furthermore, we found that the genotype with both the G allele in exon 1 and the 106 bp allele of the AT repeat in exon 4 occurred with much higher frequency in GD than NC (p<0.01), and that these polymorphisms are in linkage disequilibrium with each other. These results support the concept that CTLA-4 plays a critical role in the autoimmune process in GD, and that GD depends on multiple genetic susceptibility factors. Because the exon 1 and exon 4 polymorphisms are in strong linkage disequilibrium. It is not possible at this time to determine their unique relation to CTLA-4 function. Studies relating each polymorphism to CTLA4 function are required to determine whether one, or both, polymorphism(s) promote autoimmune disease.