We analyzed cultured cells from hyperfunctioning thyroid adenoma and its surrounding thyroid tissue from a Japanese woman and determined the nucleotide sequences of genes encoding the alpha subunit of the stimulatory G-protein 1 (G alphas) and thyrotropin (TSH) receptor in its tumor tissue. Primary culture of cells from hyperfunctioning thyroid adenoma and its surrounding thyroid tissue revealed that cAMP production was constitutively activated while intracellular Ca2+ concentration was suppressed both at the basal level and in the response to TSH stimulation in the cells from tumor tissue compared with those from non-tumor tissue. Nucleotide sequence analysis demonstrated the somatic missense mutation at codon 201 (CGT(Arg)-CAT(His)) of G alphas gene in tumor tissue but not in its surrounding tissue. No mutation was observed in the transmembrane region of TSH receptor. These results suggest that cAMP regulatory cascade is constitutively activated while phospholipase C-Ca2+ signaling cascade is suppressed in hyperfunctioning thyroid adenoma with an activating mutation of G alphas gene in the present case.