In the human thyroid, the wild-type thyrotropin receptor (TSHR) couples to adenylyl cyclase and phospholipase C and constitutively increases intracellular cAMP levels. The first human TSHR sequence submitted differs from subsequently cloned wild-type receptors by an exchange of a conserved Y residue within transmembrane domain 5 (TM5) for an H residue. We did not detect the Y601H mutant in 263 European individuals, but confirmed the homozygous occurrence of TSHR-Y601. Expression of TSHR-Y601H in COS-7 cells revealed a loss of constitutive cAMP production and selective lack of TSH-induced phosphoinositide hydrolysis, whereas agonist-induced cAMP formation remained unaltered. Analysis of several mutant receptors (Y601A, Y601D, Y601F, Y601K, Y601P, Y601S, Y601W, Y601Delta) did not show restoration of constitutive activity and dual signaling, thus suggesting a functional role of a properly spaced hydroxyl group at position 601. Molecular modeling revealed that the formation of a hydrogen bond between the hydroxyl group of Y601 in TM5 and the carbonyl oxygen of A623 in the peptide backbone of TM6 is critical for the receptor to adopt active conformations that impart wild-type signaling properties. Our findings indicate that multiple active receptor states underlie coupling of a G-protein-coupled receptor to different G-proteins.