To delineate domains of high affinity melatonin receptors that are essential for melatonin binding, we generated chimeras between the human Mel1a melatonin receptor and the melatonin-related orphan H9 receptor. The latter receptor displays no high affinity melatonin binding. The chimeric receptors were transiently expressed in COS-7 cells and analyzed by radioligand binding using 2-[126I]iodomelatonin ([125I]Mel). Replacement of individual transmembrane domains (TMs) of the Mel1a receptor by the corresponding H9 helixes revealed that TM6 plays a critical role in ligand binding. Substitution of H9-TM6 into the Mel1a receptor abolished any detectable [125I]Mel binding, whereas the remaining TMs could be readily exchanged without affecting ligand binding. Subsequent site-directed mutagenesis showed that glycine 20 in TM6 of the Mel1a receptor occupies an important position in the binding site. Thus, the mutation of glycine 20 to threonine, the corresponding H9 residue, severely reduced the receptor's affinity for melatonin. Furthermore, the double mutation of alanine 14 to cysteine and of glycine 20 to threonine in TM6 completely eliminated high affinity [125I]Mel binding. This strongly suggests that molecular modifications in TM6 that involve glycine 20 lead to steric incompatibilities in the binding pocket that prohibit high affinity melatonin binding.