Abstract: Age-related changes in levels of melatonin and 6-hydroxymelatonin sulfate and effects of dietary melatonin on their levels in different tissues were determined in mice. Levels of melatonin were highest in the serum followed by liver, kidney, cerebral cortex and heart as measured by a quantitative and sensitive enzyme-labeled immunosorbent assay (ELISA). Serum melatonin levels decreased with age, and were reduced by 80% in 27-month old mice relative to 12-month old mice. Levels of 6-hydroxymelatonin sulfate were measured independently in various tissues. Levels of the melatonin metabolite, 6-hydroxymelatonin sulfate were significantly higher than free melatonin in all tissues tested. Levels of 6-hydroxymelatonin sulfate were highest in the cerebral cortex followed by the serum, heart, kidney, and liver. In 12-month old mice 6-hydroxymelatonin sulfate concentration was approximately 1000-fold greater than that of melatonin in the cerebral cortex, it was only 3-fold greater than melatonin levels in the serum. Thus only 0.1% of total melatonin in the brain was present in the free and unconjugated form but the corresponding value for serum was 27.4%. The cerebral cortex had the highest levels of combined melatonin and 6-hydroxymelatonin sulfate than other tissue tested in control mice. There was no significant change in 6-hydroxymelatonin sulfate levels between young and old mice. There was also no age-dependent change in levels of serotonin or cortisol in the serum samples. Dietary supplementation with melatonin resulted in a significant increase in levels of melatonin in the serum and all other tissue samples tested. Thus, any age-related decline of tissue melatonin can be reversed by supplementation with dietary melatonin.