In mammals, the pineal hormone melatonin activates G protein-coupled MT(1) and MT(2) melatonin receptors. Acute exposure of recombinant MT(1) and MT(2) melatonin receptors to supraphysiological concentrations of melatonin differentially regulates these two receptors with the MT(2), but not the MT(1), exhibiting rapid desensitization and internalization. In the present study, we sought to determine whether prolonged exposure to supraphysiological and physiological concentrations of melatonin desensitized and/or internalized the MT(1) melatonin receptor. Using a Chinese hamster ovary (CHO) cell line stably expressing MT(1)-FLAG or transiently expressing MT(1)-green fluorescent protein (GFP) melatonin receptors, we found that prolonged exposure (8h) to supraphysiological concentrations of melatonin (100 nM) significantly increased the number of MT(1) melatonin receptors and decreased the affinity (K(i)) of melatonin for competition for 2-iodomelatonin. A similar treatment also desensitized the MT(1) melatonin receptor-mediated stimulation of [(35)S]GTPgammaS binding, but did not internalize the receptor. In contrast, prolonged exposure to a concentration of melatonin mimicking nocturnal levels (400 pM) did not affect the number of MT(1) melatonin receptors, the affinity for melatonin, or the functional sensitivity of the receptor. We conclude that in vivo endogenous melatonin does not significantly affect the functional sensitivity of MT(1) melatonin receptors, however, exogenous melatonin taken therapeutically at doses above physiological levels could desensitize the receptor thereby affecting physiological responses mediated following activation of MT(1) melatonin receptors.