Serum melatonin was measured by radioimmunoassay at 4-hourly intervals for 24 h in five normal men, and shown to exhibit nyctohemeral variations. The highest concentrations of serum melatonin occurred at 02.00 hours and the lowest concentrations at 14.00 hours. The synthetic enzymes for melatonin, serotonin-N-acetyl transferase (SNAT) and hydroxyindole-O-methyltransferase (HIOMT) were measured in post-mortem pineal organs from thirty-six men and women, and the activity of both enzymes was directly related to the time of death of the subject, resulting in a similar nyctohemeral curve as that observed with serum melatonin. The SNAT and HIOMT enzymes were stable when measured in pineal organs held under the usual post-mortem conditions. The observations indicated a nyctohemeral rhythm in human pineal function and suggests that any clinical interpretation of pineal dysfunction in disease states should take into consideration this normal rhythm.