Skeletal muscle contractions increase superoxide anion in skeletal muscle extracellular space. We tested the hypotheses that, (i) after an isometric contraction protocol, xanthine oxidase (XO) activity is a source of superoxide anion in the extracellular space of skeletal muscle, and (ii) the increase in XO-derived extracellular superoxide anion during contractions affects skeletal muscle contractile function. Superoxide anion was monitored in the extracellular space of mouse gastrocnemius muscles by following the reduction of cytochrome c in muscle microdialysates. A 15-min protocol of non-damaging isometric contractions increased the reduction of cytochrome c in microdialysates indicating an increase in superoxide anion. Mice treated with the xanthine oxidase inhibitor, oxypurinol, showed a smaller increase in superoxide anions in muscle microdialysates following contractions than was seen in microdialysates from muscles of vehicle-treated mice. Intact extensor digitorum longus (EDL) and soleus muscles from mice were also incubated in vitro with oxypurinol or polyethylene glycol-tagged superoxide dismutase (PEG-SOD). Oxypurinol treatment decreased the maximum tetanic force produced by both EDL and soleus muscles and PEG-SOD treatment decreased the maximum force production by the EDL muscles. Neither agent influenced the rate of decline in force production when EDL or soleus muscles were repeatedly electrically stimulated using a 5 minute fatiguing protocol (stimulation at 40Hz for 0.1 second every 5 seconds). Thus these studies indicate that XO activity contributes to the increased superoxide anion detected within the extracellular space of skeletal muscles during non-damaging contractile activity and that XO-derived superoxide anion or derivatives of this radical have a positive effect on muscle force generation during isometric contractions of mouse skeletal muscles.