It is suggested that free radical generation is a cause of muscle fatigue from in vitro studies. We examined whether hydroxyl radical would be generated in an in vivo fatiguing contraction of the diaphragm. Diaphragmatic fatigue was induced in rats by the in vivo rhythmic electrical stimulation of the diaphragm under mechanical ventilation for 30 min. The force-frequency relationship of a diaphragmatic muscle strip was assessed, and the production of hydroxyl radical in muscle strip was evaluated by measuring the metabolites of a reaction of salicylate with hydroxyl radical (2,3- and 2,5-dihydroxybenzoate [DHBA]). After fatiguing contraction, the contractile force of the diaphragm was decreased by approximately 58% at all stimulating frequencies (P < 0.01). There was more 2,3-DHBA in the fatigued muscles compared with the control muscle (219 +/- 22 ng/g tissue versus 109 +/- 17, respectively, P < 0.01) and 2,5-DHBA (198 +/- 23 ng/g tissue versus 84 +/- 14, respectively, P < 0.01). There was a significant relationship between contractile force and the sum of 2,3- and 2,5-DHBA (r = -0.71, P < 0.01). These results suggest that the development of fatigue in the diaphragm is related to the generation of hydroxyl radical.