Data regarding polymorphisms of immunoregulatory genes in polyglandular autoimmunity (PGA) are lacking. We have analyzed whether the polymorphism of the proinflammatory cytokine gene TNF-alpha; -308 and mutations of the autoimmune regulator (AIRE) gene were associated with PGA in adults. Sixty-seven patients with PGA and 209 healthy controls were genotyped by multiplex minisequencing with capillary electrophoresis on an ABI PRISM-310 genetic analyzer. HLA DRB1 typing was performed using polymerase-chain-reaction-amplified DNA hybridized with sequence-specific-oligonucleotide probes (PCR-SSO). The TNF-alpha; -308*A allele occurred more frequently in patients (0.269) than in controls (0.163, P = 0.008, P(c) = 0.016). Also, TNF-alpha; -308*A carriers were more frequent in patients than controls (47.8% vs. 31.1%, OR = 1.89, 95%CI = 1.19-3.00). The frequency of the AA genotype was increased in PGA (P = 0.014, P(c) = 0.042). PGA patients with autoimmune thyroid disease and the TNF-alpha; -308 AA genotype showed the highest prevalence of thyroid autoantibodies (TPO, P = 0.04; Tg, P = 0.003). HLA-DRB1*03 and TNF-alpha; -308*A alleles were strongly associated in patients with PGA (87.5%, P(c) < 0.00001). The AIRE R257X and 13bpdel mutations were not observed in patients with PGA. The association of TNF-alpha; -308*A with PGA might be directly or indirectly due to the association with HLA-DRB1*03.