All G protein-coupled melatonin receptors have two conserved cysteines in the interphase between transmembrane helix III and the second intracellular loop, in the region assumed important in receptor/G protein coupling. The cysteines are also potential targets of receptor S-nitrosylation. The effects of site-directed mutagenesis of these cysteines in the human MT1 melatonin receptor were investigated. The cysteines were mutated into serines either individually or as a pair and stable Chinese hamster ovary cell lines expressing the wild-type and mutant MT1 receptors were created. Receptor expression level, subcellular localization, ligand binding and G protein activation of the cell lines were analyzed. Serine substitution of C127 (Cys(3.52)) did not affect the ligand binding affinity and agonist potency but had an influence on receptor trafficking and G protein activation capacity. Serine substitution of C130 (Cys(3.55)) resulted in a decrease in the potency of melatonin to activate G proteins. When both cysteines were mutated into serines, normal MT1 receptor binding and activation were abolished. Computer modeling revealed that the mutations did not change the structure of the ligand binding pocket. Cysteine S-nitrosylation had no influence on G protein activation through MT1 receptors. Taken together, these data show that the two conserved cysteines in the end of transmembrane domain III of the MT1 melatonin receptor, especially C130 (Cys(3.55)), are needed for normal G protein activation and receptor trafficking.