The effect of photoperiod on melatonin content and the activity of the melatonin-synthesizing enzymes, namely, serotonin N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase, were investigated in the pineal gland and retina of turkeys. The birds were adapted to 3 different lighting conditions: 16L:8D (long photoperiod), 12L:12D (regular photoperiod), and 8L:16D (short photoperiod). Pineal, retinal, and plasma melatonin concentrations oscillated with a robust diurnal rhythm, with high values during darkness. The duration of elevated nocturnal melatonin levels in the turkey pineal gland, retina, and plasma changed markedly in response to the length of the dark phase, being longest during the short photoperiod with 16 h of darkness. These photoperiodic variations in melatonin synthesis appear to be driven by AANAT, because changes in the activity of this enzyme were closely correlated with changes in melatonin. By contrast, pineal and retinal hydroxyindole-O-methyltransferase activities failed to exhibit any significant 24-h variation in the different photoperiods. A marked effect of photoperiod on the level of melatonin production was also observed. Peak values of melatonin and AANAT activity in the pineal gland (but not in the retina) were highest during the long photoperiod. During the light phase, mean melatonin concentrations in the pineal gland and retina of turkeys kept under the long photoperiod were significantly higher compared with those from birds maintained under the regular and short photoperiods. In addition, mean circulating melatonin levels were lowest in the short photoperiod. Finally, the magnitude of the light-evoked suppression of nighttime pineal AANAT activity was also influenced by photoperiod, with suppression being smallest under the long photoperiod. These findings show that in the turkey, photoperiod plays an important role in regulating the melatonin signal.