The nocturnal pattern of Syrian hamster pineal melatonin synthesis is characterized by a 6-8 h lag period, followed by a late-night, short-duration peak in both N-acetyltransferase (NAT) activity and melatonin content. Administration of cycloheximide (20 mg/kg body weight) given either at the time of lights out or 4 h into the dark phase to Syrian hamsters blocked the nocturnal increase in both pineal NAT activity and melatonin content. Actinomycin D (5 mg/kg body weight) prevented the nocturnal increase in both constituents only when it was administered at darkness onset, being significantly less effective when injected after 4 h of dark exposure. Reinduction of melatonin production by isoproterenol (2 mg/kg body weight) administration to acutely light-exposed animals during late darkness was prevented by cycloheximide, but not by actinomycin D administration. The results suggest that whereas Syrian hamster pineal melatonin production requires protein synthesis both early and late in the dark phase, the transcription of a putative NAT-related mRNA, which occurs only during the early night, seems to determine the lag period in melatonin synthesis and pineal responsiveness to beta-adrenergic receptor agonist stimulation.