Melatonin MT1 and MT2 receptors are Gi protein-coupled receptors and promising therapeutic targets for a number of diseases. A proportion of G protein-coupled receptor agonists and antagonists have been classified according to their duration of action, which influences their pharmacological efficacy. However, the duration of action of melatonin agonists remains unclear. In this study, we investigated the duration of action of melatonin agonists (melatonin, 2-iodomelatonin, ramelteon, and the ramelteon metabolite M-II) at the melatonin MT1 receptor, which is more resistant to agonist-induced desensitization than the melatonin MT2 receptor. In Chinese hamster ovary cells stably expressing the human melatonin MT1 receptor, significant differences in the duration of action were observed after 2-h pretreatment with agonists followed by washout. In contrast to melatonin and M-II, the agonist activities of ramelteon and 2-iodomelatonin were persistent (i.e. inhibition of forskolin-stimulated cAMP formation and increase in ERK 1/2 phosphorylation) even after repeated washouts. Similar activities were observed for INS-1 cells endogenously expressing the rat MT1 receptor. Further, we examined potential factors linked to the duration of action. Residual activities of melatonin agonists after washout strongly correlated with their dissociation rates from the human melatonin MT1 receptor, but not their lipophilicity or extent of desensitization. These data suggest that the in vitro duration of action significantly differs between melatonin agonists and might dictate dissociation kinetics. Characterization of these in vitro properties may facilitate further in vivo study of the duration of action.