The peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha)-activated signal transduction pathway has previously been shown to stimulate mitochondrial biogenesis in skeletal muscle in response to endurance exercise. In vitro data indicate that PGC-1alpha signaling may be sensitive to reactive oxygen species (ROS) but its role in vivo is not clear. The objectives of this study were (1) to investigate whether the PGC-1alpha pathway could be activated by a single bout of anaerobic exercise in rats, wherein a major portion of ROS was generated via the cytosolic xanthine oxidase (XO), and (2) to examine whether allopurinol (ALP), a specific XO inhibitor, would attenuate PGC-1alpha expression and signaling owing to decreased ROS generation. Female Sprague-Dawley rats were randomly divided into three groups: (1) subjected to sprinting on a treadmill at 35 m/min, 15% grade, for 3 min followed by 3 min slow running at 15 m/min, 0% grade, repeated until exhaustion (88 +/- 4 min; Exer; N= 9); (2) subjected to the same exercise protocol (88 +/- 4 min) but injected with two doses of ALP (0.4 mmol/kg, ip) 24 and 1 h before the experiment (Exer+ ALP; N= 9); and (3) rested control (C; N= 9). Exercise increased XO activity and ROS generation in the Exer rat vastus lateralis muscle (P< 0.05), whereas the Exer+ ALP group displayed only 7% XO activity and similar ROS level compared with the C group. PGC-1alpha protein content showed a 5.6-fold increase (P< 0.01) in Exer vs C, along with a 200% (P< 0.01) increase in both nuclear respiratory factor (NRF)-1 and mitochondrial transcription factor A (Tfam) content. ALP treatment decreased PGC-1alpha, NRF-1, and Tfam levels by 45, 19, and 20% (P< 0.05), respectively. Exercise doubled the content of the phosphorylated cAMP-responsive element-binding protein in the Exer group (P< 0.01) and tripled phosphorylated p38 mitogen-activated protein kinase (P< 0.01), whereas these effects were reduced by 60 and 30% (P< 0.01, P< 0.05), respectively, in Exer+ ALP rats. Nuclear factor-kappaB binding and phospho-IkappaB content were also increased in Exer rats (P< 0.01) and these increases were abolished by ALP treatment. The data indicate that contraction-activated PGC-1alpha signaling pathways in skeletal muscle are redox sensitive and that nonmitochondrial ROS play an important role in stimulating mitochondrial biogenesis.