The neurohormone melatonin is the central switch of the circadian rhythm and presumably exerts its activities through a series of receptors among which MT 1 and MT 2 have been widely studied. The third binding site of melatonin, MT 3 , has been recently characterized as a melatonin-sensitive form of the quinone reductase 2 (QR 2 , EC 1.6.99.2). In the present work, we showed that the binding of melatonin at MT 3 /QR 2 was better described with 2-[ 125 I]-iodomethoxy-carbonylamino-N-acetyltryptamine (2-[ 125 I]-I-MCA-NAT) and, most importantly, that it was measurable at 20° while it has been initially described and thoroughly studied using 2-[ 125 I]-iodomelatonin at 4°. Under these novel conditions, binding to MT 3 could be traced without cross-reactivity with MT 1 and MT 2 receptors and, moreover, under conditions similar to those used to measure MT 3 /QR 2 catalytic activity. The pharmacology established here on hamster kidney samples using the reference compounds remained essentially as already described using other experimental conditions. A new series of compounds with nanomolar affinity for the MT 3 binding site and a high MT 3 selectivity versus MT 1 and MT 2 is reported. In addition, we further document the MT 3 /QR 2 binding site by demonstrating that it was widely distributed among mammals, although inter-species and inter-tissues differences exist. The present report details new experimental conditions for the pharmacological study of melatonin-sensitive QR 2 isoforms, and suggests that, in addition to an already demonstrated inter-species difference, inter-tissues differences in QR 2 sensitivity to melatonin may exist in primates and, therefore, represent an original and interesting route of investigation on the effect of melatonin on MT 3 /QR 2 .