These studies investigated the characteristics and regional distribution of 2-[125I]iodomelatonin binding in Djungarian hamster brain. The results showed that 2-[125I]iodomelatonin labels two types of binding sites, which resemble the ML-1 and ML-2 melatonin subtypes previously described in other tissues. The 2-[125I]iodomelatonin binding site identified in whole brain membranes has a nanomolar affinity (Kd = 1.48 +/- 0.26 nM) and biochemical and pharmacological characteristics identical to those of the ML-2 site of Syrian hamster whole brain. The 2-[125I]iodomelatonin site in the hypothalamus has a picomolar affinity (Kd = 43.4 +/- 5.1 pM) and resembles the ML-1 site of chicken retina. The localization of 2-[125I]iodomelatonin labeling in autoradiographic studies of the Djungarian hamster brain includes the suprachiasmatic nuclei, the median eminence, the reuniens nucleus, and the paraventricular nucleus of the thalamus.