Formation of the pineal gland hormone melatonin increases markedly at night in response to light-dark environmental alterations. Melatonin is synthesized from serotonin by an initial N-acetylation followed by methylation of the 5-hydroxy moiety by hydroxyindole-O-methyltransferase. Serotonin N-acetyltransferase (NAT; EC22.214.171.124), which catalyses the first reaction, is the rate-limiting enzyme in this process, and its activity increases dramatically with the onset of darkness. Because melatonin may play important biological roles in reproduction, ageing and sleep, understanding the molecular factors that regulate NAT is of particular importance. To identify proteins that regulate light-dark variations in pineal function, we used a subtractive hybridization technique based on the polymerase chain reaction (PCR) to isolate rat pineal gland messages that are differentially expressed by day and night. Here we report the molecular cloning of NAT and dramatic diurnal variations in its transcription. Independently, Klein and associates have cloned NAT from sheep pineal glands.