Exhaustive exercise generates excessive amounts of oxidative free radicals (Sastre et al, 1992) that overwhelm cellular antioxidant defences and may cause tissue damage (Gomez-Cabrera et al, 2003). They may, however, constitute signals to regulate muscle cell function. Accordingly, there is considerable interest in the potential of these mediators to regulate muscle adaptation to exercise. This is one of the oldest postulates in the field, dating back to the suggestion that free radicals produced in exercising muscle might stimulate mitochondrial biogenesis (Davies et al, 1981) and the expression of genes for antioxidant enzymes. Moderate exercise increases lifespan in humans but this may be due to other healthy habits of the exercising population. The aim of this study is to elucidate the role of the free radicals generated in moderate physical exercise, in the expression of antioxidant genes and of transcription factors for mitochondrial biogenesis. Twenty male wistar rats were randomly divided into four groups: sedentary controls (n=5), exercised (n=5), exercised treated with 500 mg/Kg of vitamin C (n=5) and exercised treated with 32 mg/Kg of allopurinol (n=5). Allopurinol acts as an antioxidant because it inhibits xanthine oxidase, an important generator of free radicals in exercise (Gomez-Cabrera et al, 2003). The experimental protocol was approved by the Committee on Ethics in Research of the Faculty of Medicine Valencia. Where indicated animals were subjected to moderate exercise training (10, 17, 24 and 26 m/min for 5, 10, 10 and 20 min. each, five days a week during three weeks). Rats were anesthetized with 50 mg/kg sodium pentobarbithal by i.p. injection and gastrocnemius muscle was obtained by quick removal. Rats were killed by an ovedose of the anesthetic. Our results, using quantitative Real-Time RT-PCR, show that moderate exercise significantly up-regulates the expression of antioxidant enzymes associated with longevity, such as Mn-SOD (3.5-fold change, P<0.0001) and GPx (3.4-fold change, P<0.0001). We also found that moderate exercise up-regulated the expression of NRF-1 (14-fold change, P<0.0001) that is a key transcriptional activator of nuclear genes encoding mitochondrial enzymes and Tfam (30-fold change, P<0.0001), which stimulates mitochondrial DNA transcription and replication. However, supplementation with vitamin C or allopurinol during training prevented all of these adaptations. Significance was determined by ANOVA followed by Tukey test relative to sedentary controls after normalization to GAPDH. We conclude that oral antioxidant supplementation is very likely to be useful before competition when exercise is likely to be exhaustive, and damaging (Gomez-Cabrera et al, 2003), but not when training.