MELATONIN is synthesized in the chicken retina under the influence of a circadian clock, which also regulates the expression of tryptophan hydroxylase (TPH) and serotonin N-acetyltransferase (AA-NAT). In order to examine the role of substrate supply in the rhythmic synthesis of melatonin in chicken retina, tryptophan and 5-hydroxytryptophan were administered day and night in light or darkness. When administered systemically at night in darkness, 5-hydroxytryptophan, but not tryptophan, dramatically stimulates melatonin levels in the chick retina in a dose-dependent manner. Intraocular administration of 5-hydroxytryptophan also increases melatonin levels locally, indicating a retinal site of action of the serotonin precursor. The effect of 5-hydroxytryptophan is much greater at night, when TPH and AA-NAT activities are high, than during the day, when the enzyme activities are low. Similarly, unexpected light exposure at night, which inactivates AA-NAT, significantly reduces the ability of 5-hydroxytryptophan to increase retinal melatonin levels. The results suggest that TPH, but not AA-NAT or other enzymes in the melatonin biosynthetic pathway, is saturated with substratein sit u. The rate of melatonin production appears to be a function of the concentration of serotonin, which is regulated by TPH, and by the level of activity of AA-NAT.