Both melatonin and leptin show a circadian variation in circulating levels and participate in energy metabolism. An interrelationship between these two hormones has thus been proposed. In addition, melatonin has been shown to be capable of influencing circulating leptin concentration. However, whether melatonin will increase or decrease leptin production is still uncertain. This study was undertaken to examine the effect of melatonin on leptin production using male C57BL/6 adult mice treated with or without daily melatonin supplements (10 mug/mL) in drinking water for 1 month. In addition, in vitro experiments using adipose tissue fragments derived from epididymal fat pads of adult mice incubated with or without melatonin (1 nM) administration were also conducted. The results showed that melatonin-supplemented mice had significantly higher plasma leptin levels than control mice. However, melatonin incubation did not cause any marked changes in the amount of leptin secreted from adipose tissue fragments. Our findings from this study indicate that melatonin does not affect leptin secretion via mouse adipose tissue. Nevertheless, melatonin could still influence leptinemia indirectly via regulatory effects in intact animals.