The concentration of melatonin shows a marked diurnal rhythm in the pineal gland and in blood with high levels normally occurring during dark and low levels during light. Synthesis in the pineal gland is controlled by a noradrenergic mechanism regulated by an indirect neural pathway from the eye. The pineal enzymes N-acetyltransferase and hydroxyindole-O-methyltransferase are activated when noradrenaline is released during darkness resulting in synthesis and release of melatonin. Hydroxyindole-O-methyltransferase has also been identified in the retina, Harderian gland and gut. We have demonstrated melatonin in these structures. Melatonin has also been localized in the optic chiasm, suprachiasmatic nucleus and in the palatine salivary gland but it is not known whether it is synthesized in these locations or whether extrapineal melatonin enters the circulation.In both diurnal and nocturnal species melatonin in circulation shows peak levels in the dark and low levels in the light. There is evidence for an endogenous rhythm generator responsible for synchronizing rhythms including the melatonin rhythm at approximately a 24 hour interval which can be cued by light. The onset of light rather than the onset of darkness appears to act as the cue or Zeitgeber. If the hamster or rat is exposed to 1½ or 2 hours of light in the 24 hour day, pineal and serum melatonin rhythms are synchronized in phase with light onset rather than onset of darkness. The melatonin rhythm can be dissociated from the adrenal rhythm. Restricting feeding to early hours of light in the rat will cause a shift in the corticosterone rhythm without altering the rhythm in plasma or pineal melatonin. Increasing the length of darkness does not necessarily produce a longer peak of melatonin in blood. In the hamster or rat, exposed to a shortened photoperiod with only 1½ or 2 hours of light per day both the pineal and serum melatonin rhythms are generally unchanged. There appears to be feedback regulation of circulating melatonin. In rats immunized against melatonin to cause a lowering of circulating melatonin, pineal content is increased.High affinity binding of melatonin to brain cytosol and membrane as well as to testicular and ovarian cytosol has been described and may be indicative of receptors through which melatonin produces its central and peripheral effects. We have found cytosol binding in a variety of human, bovine and rat brain regions with an affinity approximately three times that of the reported membrane binding. Melatonin binding has also been defined by immunofluorescence in the pineal gland. This binding has a diurnal rhythm differing from that of melatonin and has an affinity constant in the nanomolar range.