AbstractSoybean lipoxygenase‐mediated cooxidation of reduced glutathione (GSH) and concomitant superoxide generation was examined. The oxidation of GSH was dependent on the concentration of linoleic acid (LA), GSH, and the enzyme. The optimal conditions to observe maximal enzyme velocity included the presence of 0.42 mM LA, 2 mM GSH, and 50 pmole of enzyme/mL. The GSH oxidation was linear up to 10 minutes and exhibited a pH optimum of 9.0. The reaction displayed aKmof 1.49 mM for GSH andVmaxof 1.35 ± 0.02 μmoles/min/nmole of enzyme. Besides LA, arachidonic and γ‐linolenic acids also supported the lipoxygenase‐mediated GSH oxidation. Hydrogen peroxide and 13‐hydroperoxylinoleic acid supported GSH cooxidation, but to a very limited extent. Oxidized glutathione (GSSG) was identified as the major product of the reaction based on the depletion of nicotinamide‐adenine dinucleotide 3′‐phosphate (NADPH) in the presence of glutathione reductase. The GSH oxidation was accompanied by the reduction of ferricytochromec, which can be completely abolished by superoxide dismutase (SOD), suggesting the generation of superoxide anion radicals. Under optimal conditions, the rate of superoxide generation (measured as the SOD‐inhibitable reduction of ferricytochromec) was 10 ± 1.0 nmole/min/nmole of enzyme. These results clearly suggest that lipoxygenase is capable of oxidizing GSH to GSSG and simultaneously generating superoxide anion radicals, which may contribute to oxidative stress in cells under