We have discovered two somatic mutations in the VI transmembrane domain of the thyrotropin receptor gene in thyroid hyperfunctioning adenomas. The mutated amino acid residues are phenylalanine 631 (to cysteine) and threonine 632 (to isoleucine). Cloning and expression of the mutated versions of the receptor in COS cells increased significantly the basal and the TSH-induced cAMP levels compared to the wild type receptor. Moreover, the expression of a reporter gene under the control of the cAMP-inducible promoter, was likewise constitutively activated in cells expressing the 631 and 632 TSH receptor mutants relative to the wild type. These data indicate that the VI transmembrane segment in the TSH receptor and presumably in the other G-protein coupled receptors is a critical domain for the activation of G-protein signalling and that the mutations described here may be the cause of the thyroid hyperfunctioning adenoma.