Melatonin is a widely distributed hormone that regulates several major physiological processes, including the circadian rhythm and seasonal adaptation. The two subtypes of mammalian G protein-coupled melatonin receptors are primarily responsible for mediating the actions of melatonin. Because synthetic melatonin agonists have considerable therapeutic potentials in modulating insomnia and circadian- related sleep disorders, it is highly desirable to develop subtype-selective melatoninergic compounds. The pharmacological potencies of a series of substituted N-[3-(3-methoxyphenyl)propyl] amides towards human melatonin MT1 and MT2 receptors were evaluated by the FLIPR high-throughput screening assay, whilst their subtype-selectivity was subsequently verified with ERK phosphorylation and cAMP assays. Structure-activity relationship analysis of highly potent subtype-selective ligands (MT2 EC50 10-90 pM) revealed that a benzyloxyl substituent incorporated at C6 position of the 3-methoxyphenyl ring dramatically enhanced the MT2 potency and at the same time decreased MT1 potency. Incorporation of structural moieties conferring the subtype selectivity produced several extremely potent MT2-selective ligands. The most potent subtype-selective ligand, 2q had a substantially higher potency for MT2 receptor than melatonin for elevation of [Ca2+]i and inhibition of forskolin-elevated cAMP. Representative MT2-selective ligands also induced ERK phosphorylation in both recombinant and native cell lines, and no cross-reactivity to 17 other GPCRs could be detected. These ligands represent invaluable tools for delineating the functional roles of distinct melatonin receptor subtypes and are viable candidates for drug development.