ABSTRACTMelatonin is a neurohormone released in a circadian manner with peak levels during the night. In mammals, melatonin mediates its effects mainly through G protein-coupled MT1 and MT2 receptors. Drugs acting on melatonin receptors are indicated for circadian rhythm- and sleep-related disorders and major depression. Pharmacological tools to study the activation of these receptors with high temporal resolution are lacking. Here, we synthesized a family of light-activatable caged melatonin compounds by covalently attaching o-nitrobenzyl (o-NB) or coumarin photocleavable groups to the N-position of melatonin. All caged compounds showed the expected decrease in binding affinity for MT1 and MT2. Among them, the o-NB derivative MCS-0382 showed the best uncaging and biological properties upon light activation with a significant increase (2.5 log left-shift) in affinity and potency in melatonin receptor binding and signaling, respectively. Generation of biologically active melatonin from MCS-0382 was further demonstrated by its ability to modulate the excitation of SCN neurons in rat brain slices. MCS-0382 is now available to study melatonin effects in a temporally controlled manner in cellular and physiological settings.