Superoxide anion radical (O₂^•−) is released from skeletal muscle at rest and is particularly elevated during conditions of heat stress (42°C). Previous studies have shown that in isolated rat diaphragm O₂^•− release is not dependent on mitochondrial electron transport, reduced NADP oxidase activity, or the integrity of membrane anion channels. This study hypothesized that O₂^•− release, as measured by cytochrome c reduction, is linked to metabolism of arachidonic acid. Phospholipase A₂ inhibition with manoalide significantly decreased O₂^•− release. In downstream pathways, neither the blockage of cyclooxygenase with indomethacin nor the inhibition of cytochrome P-450-dependent monooxygenase with SKF-525A decreased O₂^•− release. However, lipoxygenase (LOX) inhibition with general LOX blockers 5,8,11,14-eicosatetraynoic acid and cinnamyl-3,4-dihydroxy-α-cyanocinnamate greatly attenuated the signal. Furthermore, the specific 5-LOX inhibitor diethylcarbamazine also significantly decreased O₂^•− release. Immunohistochemistry localized 5- and 12-LOX to the cytosol and sarcolemma of muscle cells. Confocal studies, using the O₂^•−-sensitive fluorescent indicator hydroethidine, demonstrated that LOX inhibition had no significant influence on intracellular O₂^•− formation. When compared with the cytochrome c results, this indicates that intra- and extracellular O₂^•− must arise from different sources. These data show for the first time that arachidonic acid metabolism through LOX activity, is a major source of extracellular O₂^•− release in skeletal muscle.