We evaluated the role of reactive oxygen species (ROS) for the contraction induced increase in expression of PGC-1alpha, HKII and UCP3 mRNA. Rat skeletal muscle cells were subjected to acute or repeated electrostimulation in the presence and absence of antioxidants. Contraction of muscle cells lead to an increased H2O2 formation, as measured by oxidation of H2HFF. Acute contraction of the muscle cells lead to a transient increase in PGC-1alpha and UCP3 mRNA by 172 and 65%, respectively (p<0.05), whereas this increase was absent in the presence of antioxidants. Repeated contraction sessions induced a sustained elevation in PGC-1alpha and UCP3 mRNA and a transient increase in HKII (p<0.05) and this effect was not present with treatment of cells with either an antioxidant cocktail or with GPX+GSH. Incubation of cells for 10 days with ROS produced by xanthine oxidase/xanthine increased the level of PGC-1alpha, HKII and UCP3 mRNA by 175, 58 and 115%, respectively (p<0.05). A 10-day incubation of cells with antioxidants was found to have no effect on the basal mRNA content (p>0.05). The present data demonstrate that contraction of skeletal muscle cells leads to an enhanced formation of ROS and an elevation in PGC-1alpha, UCP3 and HKII mRNA content which is abolished in the presence of antioxidants, suggesting that ROS are of importance for the contraction induced increase in expression of these genes in skeletal muscle.