It is well documented that regular physical activity and increased levels of melatonin may enhance the blood antioxidant system. The aim of the present study was to evaluate the antioxidant defense mechanisms, in particular, the antioxidant potential of melatonin in groups of elderly men (mean age 70 years), marked of on the basis of habitual physical activity level. To estimate the physical activity of the study population, weekly energy expenditure was measured in each subject with a Caltrac accelerometer. The study population was divided into two groups (31 subjects in each group) according to the criteria proposed by Bauman: less active (energy expenditure below 1600 kcal per week) and more active (energy expenditure 1600 kcal per week). Venous blood samples were taken from each subject for assessment of plasma total antioxidant status (TAS), thiobarbituric acid reactive substances (TBARS) concentrations and melatonin (MLT) levels. The reduced glutathione (GSH) content and glutathione peroxidase (GPx) activity in hemolyzed red blood cells were also measured. A significant (p < 0.05), inverse Spearman correlation between MLT concentrations in plasma and physical activity levels was observed in the group of less active men. In more active subjects, a positive correlation between GSH and TAS levels (p < 0.05) and inverse correlations between GSH and TBARS concentrations (p < 0.05), TAS and TBARS levels (p < 0.01) and between TAS and MLT levels (p < 0.001) were noted. The results of the present study support the view that melatonin plays an important role in the blood antioxidant defense system in elderly men and indicate that habitual physical activity may have an influence on the plasma levels of melatonin.