Constitutively activating mutations of the thyrotropin (TSH) receptor have been identified as a molecular cause of toxic adenomas, nonautoimmune familial hyperthyroidism, and sporadic congenital hyperthyroidism. By analyzing genomic DNA from a toxic adenoma, we detected a novel somatic mutation in codon 601, tyrosine to asparagine (Y601N), a residue located in the carboxyterminal part of the fifth transmembrane helix. This codon is also notable for the presence of a polymorphic variant, Y601H. These two naturally occurring substitutions (Y601N and Y601H) were analyzed together with an artificial mutation, Y601F, to study the role of this residue for receptor function further. Transient transfection assays revealed that the Y601N mutation results in constitutive activation of the cyclic adenosine monophosphate (cAMP) pathway, but that it is unable to couple to G_q/11. Y601H and Y601F do not display basal activity while retaining responsiveness to TSH, but also lose the ability to induce inositol phosphate accumulation in response to TSH. These studies define Y601N as a mutation that selectively activates the cAMP pathway, and they confirm that Y601H is not a silent polymorphism. In conclusion, residue Y601 has an important role for the characteristic constitutive basal activity of the TSH receptor and coupling to G_q/11.