Pineal N-acetyltransferase (NAT) activity and radioimmunoassayable levels of melatonin were compared in 2-month-old (young), 12-month-old (middle-aged), and 29-month-old (old) female rats killed at 1600 h (during the light) and at 2300 h (4 h after darkness onset) and 0100 h (6 h after darkness onset). During the light period, NAT levels were equivalent in pineals from each age group. With the onset of darkness NAT levels rose sharply and were again equivalent in all groups at 2300 h. At 0100 h pineal NAT values in the old rats were lower than in the other two groups. Melatonin values were low in pineal glands of all animals killed at 1600 h in light. By 4 h after darkness onset pineal melatonin content in the young rats had increased 12-fold compared to a 6-fold rise in the old animals. Melatonin levels in the middle aged rats were intermediate between the other two groups. Similar relationships were observed in the rats killed at 0100 h. By this time the young rats had melatonin levels 17 times higher than during the day while the increase in the old rats was only 7-fold; 12-month-old rats again had intermediate levels. The finding show a marked reduction in pineal melatonin with aging in female rats.