Osteoporosis is associated with many etiological causes such as nutrition, cytokines, hormones, and aging. Recently, reactive oxygen species (ROS) are considered to be responsible for the aging process and osteoporosis. We investigated the relationship between ROS and bone metabolism in young female and postmenopausal rats, by using dietary iron overload and several indices including bone metabolic markers, oxidative stress and antioxidant markers, and cytokines. Postmenopausal rats exhibited significant decreases in serum alkaline phosphatase activity and the level of osteocalcin as bone formation markers compared with young female rats; however, urinary excretion of deoxypyridinoline, a bone resorption marker, did not change. On the other hand, a 5% iron lactate diet for 4 weeks in postmenopausal rats led to significantly increased excretion of urinary deoxypyridinoline and 8-hydroxy-2'-deoxyguanosine (8-OHdG) but not serum alkaline phosphatase activity. Interestingly, the diet induced significant increases of serum osteopontin and TGF-beta1, augumenting osteoclast-mediated bone resorption through the RANK/RANKL pathway [J. Clin. Invest. 112 (2003) 181]. TGF-beta1 showed a negative correlation with serum glutathione peroxidase (GPx) activity (r = -0.674, P < 0.003), but a positive correlation with the serum iron level (r = 0.836, P < 0.0001). Taken together, these results suggest for the first time that oxidative stress could be involved in the pathogenesis of metabolic bone diseases such as osteoporosis as demonstrated by analysis of the relationship between bone metabolism and oxidative stress.