We used mouse soleus in vitro (n = 30) and canine gastrocnemius-plantaris preparations (n = 20) pump-perfused at the animal's blood pressure to establish if free radicals contribute to fatigue in oxidative skeletal muscle. The soleus from each leg contracted for 200 ms (70 Hz) once every minute for 60 min in Hepes buffer gassed with 100% oxygen at 27 degrees C. When contracting in Hepes alone, both muscles fatigued at 0.9 mN/mm2.min over the 60 min. The addition of purines to the bath increased the rate to 1.4 mN/mm2.min and the addition of xanthine oxidase to generate free radicals increased the rate again to 1.9 mN/mm2.min. Thus free radicals appeared to attenuate oxidative skeletal muscle function. Each canine muscle contracted isometrically at 4 Hz for 30 min and then rested for 45 min before contracting for a second 30 min at 4 Hz. In each experiment, we infused saline at 0.76 mL/min into resting muscle and at 1.91 mL/min during the first contraction period. During the remainder of the experiment, we infused, at the same rates, saline (n = 4), 10 microM dimethyl sulfoxide (DMSO) (n = 4) to identify the effect of scavenging hydroxyl radicals, 1 mM allopurinol to establish the effect of blocking xanthine oxidase (n = 4), or 200 microM desferoxamine to determine the effect of chelating iron (n = 4). With saline, the fatigue rate over the 30 min of contractions increased from 5.0 +/- 0.2 to 6.3 +/- 0.5 N/kg.min from the first to the second stimulation period. The fatigue rate was slower in the second period with each of the three experimental substances (DMSO, 5.9 +/- 0.8 to 3.2 +/- 0.3; allopurinol, 7.3 +/- 1.1 to 4.6 +/- 0.6; desferoxamine, 6.8 +/- 0.8 to 4.4 +/- 0.8 N/kg.min). The fatigue rate was the same as control when DMSO was infused only during the second contraction period. Therefore, free radicals appeared to contribute to fatigue in oxidative skeletal muscle.