Free radicals have been theorized to play a causative role in the normal aging process. To date, methods used to detect oxidative stress in aged experimental animals have only detected 2- to 3-fold differences or less between young and aged animals. Measurement of F-2-isoprostanes has emerged as probably the most reliable approach to assess oxidative stress status in vivo. Therefore, we measured levels of F-2-isoprostanes free in plasma and levels esterified in plasma lipids in young rats (3-4 months of age) and aged rats (22-24 months of age). Plasma concentrations of free F-2-isoprostanes were increased dramatically by a mean of 20.3-fold (range 4.3 to 42.9-fold) and levels esterified in plasma lipids were also strikingly increased by a mean of 29.9-fold (range 15.8 to 50.0-fold). These findings unveil profound oxidative stress in aged rats which adds considerable support for the free radical theory of aging.