Recent reports have demonstrated that superoxide is released by the contracting diaphragm. Moreover, extracellular scavengers of superoxide (i.e., exogenously administered superoxide dismutase) reduce diaphragm fatigue rate, arguing that superoxide released from contracting muscles may have functionally significant effects. The mechanism by which free radical formation and release occurs has not, however, been determined, and all past studies of this phenomenon have been conducted at a single muscle length (the length of maximum force generation, Lo) and at a single level of carbon dioxide. The purpose of the present study was twofold: (1) to examine the effect of blockade of two free radical-generating pathways (i.e., to block cyclooxygenase with indomethacin and xanthine oxidase with oxypurinol) on superoxide release by the contracting diaphragm, and (2) to examine the effect of altering muscle length, carbon dioxide levels, and stimulation frequency on superoxide release during contraction. Studies were performed using an isolated, arterially perfused, rat diaphragm preparation in which superoxide release was assessed in real time by measuring arteriovenous cytochrome c reduction gradients across this muscle. We found that superoxide release during contraction was: (1) not altered by indomethacin administration, (2) partially reduced by oxypurinol administration, (3) reduced by decreasing muscle length, (4) reduced by increasing carbon dioxide concentrations, and (5) reduced by decreasing stimulation frequency. The first two findings indicate that xanthine oxidase pathways contribute to free radical formation under these circumstances but cyclooxygenase does not. The last three findings suggest that these common physiologic alterations have significant effects on free radical release by contracting muscle.