OBJECTIVES: In the present paper the circadian variation and age differences in human leukocyte reactive oxygen species (ROS) levels and plasma total antioxidant status were studied. Different ROS levels could influence disease during ageing and at different times of the day/night. METHODS: Unstimulated and stimulated (12-O-tetradecanoyl-phorbol-13-acetate (TPA)) blood samples were analysed by flow cytometry using dihydroethidium (DHE) and dihydrorhodamine 123 (DHR) as probes. 60 healthy individuals were divided into five equal groups ((I) 1-2 days post partum, (II) 20 +/- 2 years, (III) 40 +/- 2 years, (IV) 60 +/- 2 years and (V) 80 +/- 2 years) and 6 healthy volunteers were followed with blood samples at 3 hour intervals for 24 hours. RESULTS: We observed a significant peak of ROS levels in both monocytes and granulocytes at 6 pm and 3 am using DHE as probe. DHR showed in principle the same pattern. The monocytes (unstimulated and stimulated) and unstimulated granulocytes of the newborn group showed higher ROS values than all the other groups, whereas the groups of adults did not differ from each other. The plasma total antioxidant status was significantly higher in the newborn group and showed no circadian variation. CONCLUSIONS: The present data show that there is a circadian variation of ROS production in leukocytes that might possibly influence the occurrence of cardiovascular incidents like myocardial infarction. No increase in ROS production was found in healthy elderly compared to younger individuals. The increased ROS levels and antioxidant status of newborns might influence neonate immunity and play a part in cell signalling and development.