Melatonin, a suspected hormone of the pineal organ, was extracted from chicken serum and quantified by the Rana pipiens bioassay. When chickens were housed in an alternating light-dark photoperiod, serum melatonin was usually not detected during the light phase. Serum melatonin rose within 1 hr after the lights were extinguished, plateaued around midnight and fell before the lights were turned on. Serum melatonin was not detected at any time of day in pinealectomized chickens, suggesting that the pineal is neccessary for, and may be the source of, circulating melatonin.