We determined melatonin reference limits in blood serum of 129 healthy volunteers, aged 21 to 70 years, using enzyme-linked immunosorbent assay (IBL ELISA kit, Hamburg, Germany). At 3:00 AM, in almost full dark, the reference limits were found to be 18.3-134 pg/ml in males, 19.0-197 pg/ml in females and 18.5-180 pg/ml in the whole group. At 8:00 AM, they were statistically lower (5.63-61.8, 3.30-93.2 and 3.83-80.4 pg/ml respectively) possibly due to the light.