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In the present paper, binding of melatonin to purified cell nuclei from harderian glands of male and female hamsters was assessed. Binding of 125I-melatonin to cell nuclei fulfills the criteria for binding to a receptor site. Binding kinetics exhibit properties such as dependence on time and temperature as well as reversibility, saturability, high affinity and specificity. The dissociation constants (KD) and the number of binding sites (Bmax) for the binding of 125I-melatonin to harderian gland nuclei were 260 ± 56 pM and 12.2 ± 0.8 fmol/mg protein in male glands, and 280 ± 43 pM and 9.8 ± 0.6 fmol/mg protein in female glands, respectively. Competition experiments showed IC50 values for melatonin of 250 ± 45 pM and 290 ± 68 pM in male and female glands, respectively. Other indoleamines such as N-acetylserotonine and 5-metoxytryptamine showed IC50 values in the micromolar range, suggesting that the binding sites are specific for melatonin. Hill analyses of the data show nH values of 0.96–0.98, suggesting the existence of a single class of binding sites. These data indicate that specific 125I-melatonin binding sites exist in the cell nuclei of Harderian glands in male as well as in female hamsters, without significant differences between them. The KD and Bmax values obtained from the binding in both sexes correlates well with the concentration of melatonin described in these respective Harderian glands. It is hypothesized that the nuclear binding sites of melatonin here described could be a physiological melatonin receptor, which may be involved in the genomic-dependent antioxidant effects of melatonin on hamster Harderian glands elsewhere reported.