Somatic mutations of the thyrotropin receptor (TSHR) gene and the gene encoding the alpha subunit of the stimulatory GTP-binding protein (Gsalpha) are the main cause for autonomously functioning thyroid nodules (AFTN) in iodine-deficient regions of the world. In iodine-sufficient regions, including Japan, the genetic relevance of AFTN is unclear. In a series of 45 Japanese subjects with AFTN, exons 9 and 10 of the TSHR and exons 7-10 of Gsalpha , where the activating mutations have been found, were analyzed using direct sequencing. We found 29 somatic mutations: 22 in the TSHR gene and 7 in the Gsalpha gene. The most frequent mutation in TSHR was Met453Thr (10 cases), followed by clustered residues from codons 630 through 633 on TSHR (7 cases). Mutations of Gsalpha were detected at codon 201 in 5 cases and at codon 227 in 2 cases. No patients had coexistent TSHR and Gsalpha mutations in the same nodule. All mutated residues but one, which was deleted at codon 403 on the TSHR gene, are constitutively active. The prevalences of a germline polymorphism of Asp727Glu on the TSHR gene and incidental papillary thyroid carcinoma in thyroid surgical specimens were similar to those reported in other studies. In the present study, more than half of the cases with AFTN had a somatic activating mutation either of the TSHR or Gsalpha gene, despite their high iodine intake.