This study was undertaken to investigate the effects of exercise, dietary restriction (DR) and aging on the formation of reactive oxidant species (ROS), antioxidant defenses, and membrane fluidity. Test were performed on hepatic microsomes, mitochondria, and cytosol from 9- and 20-month-old male Fischer 344 rats, which were divided into four groups: ad libitum fed, sedentary (AS); restricted, sedentary (RS); ad libitum fed, exercised (AE); and restricted, exercised (RE). Results show that both exercise and DR suppressed microsomal ROS production, but not mitochondrial ROS production, which increased with age in all groups. Exercise and DR increased catalase and glutathione peroxidase (GSH-Px) activities and maintained cytosolic ascorbic acid concentration at high levels. Exercise led to significantly higher levels of cytosolic glutathione (GSH). Activity of cytosolic superoxide dismutase (SOD) remained unchanged, whereas glutathione-s-transferase (GST) activity significantly increased with DR. The fluidity of the mitochondrial membrane from exercised and DR rats deteriorated less with age than the membrane from AS rats. Exercise alone was found to improve fluidity, but was more effective when coupled with DR. These results suggest for the first time that the combination of exercise training and DR is the most effective means of preserving membrane fluidity and suppressing microsomal ROS production.