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In vitro autoradiography with ¹²⁵I-labeled melatonin was used to examine melatonin binding sites in human hypothalamus. Specific ¹²⁵I-labeled melatonin binding was localized to the suprachiasmatic nuclei, the site of a putative biological clock, and was not apparent in other hypothalamic regions. Specific ¹²⁵I-labeled melatonin binding was consistently found in the suprachiasmatic nuclei of hypothalami from adults and fetuses. Densitometric analysis of competition experiments with varying concentrations of melatonin showed monophasic competition curves, with comparable half-maximal inhibition values for the suprachiasmatic nuclei of adults (150 picomolar) and fetuses (110 picomolar). Micromolar concentrations of the melatonin agonist 6-chloromelatonin completely inhibited specific ¹²⁵I-labeled melatonin binding, whereas the same concentrations of serotonin and norepinephrine caused only a partial …