Melatonin has been shown to be an effective antioxidant in a number of experimental models both in vitro and in vivo. However, limited information is available on monitoring the effect of melatonin on the antioxidant enzyme status in the liver in single circadian rhythm. To monitor the effect of melatonin on antioxidant enzyme activity, we used Swiss albino inbred male mice as a model system. The crude enzyme fractions were prepared from freshly isolated livers at 0, 2, and 24 hours as light cycle and 4, 8, and 12 hours as dark cycle specimens, post injection of melatonin. The injection of melatonin resulted in a statistically significant decrease in activity of SOD compared to control group (p<0.01). However, harvesting time did not affect SOD levels significantly. The activity of CAT was significantly decreased by melatonin and this decrease was clearly related to the time of harvest (p<0.01). Inhibition on SOD activity started at the beginning of the dark cycle of the circadian rhythm, while CAT activity started to decrease at that point. The inhibition of CAT reached its maximum at 8 hour post injection. After 12 hour post injection, the beginning of the light cycle period; CAT activity turned back to the control level. We concluded that based on control data, internal melatonin release increases SOD and CAT activity. The external melatonin treatment directly inhibits mostly •OH radicals and H2O2 as well as singlet oxygen (1O2).