The generation of oxygenated products from arachidonic acid and docosahexaenoic acid by the n-9 lipoxygenase of trout gill was monitored as a function of substrate concentration and added glutathione. In the absence of added glutathione up to 50% of the substrate consumed by the lipoxygenase was ultimately converted non-enzymatically to trihydroxy derivatives of the initial n-9 hydroperoxide enzyme product. The presence of added glutathione progressively increased conversion of the respective fatty acid hydroperoxides to the n-9 monohydroxy derivatives of arachidonic and docosahexaenoic acids while concomitantly decreasing the yield of trihydroxy derivatives, consistent with its role as a cosubstrate in the peroxidase reaction. The stability and net turnover of the lipoxygenase were also significantly improved by the addition of glutathione. The relative distribution of monohydroxy and trihydroxy products from either arachidonic acid or docosahexaenoic acid were similarly affected and equally sensitive to the glutathione concentration. These data suggest that in animals, the hydroperoxides of n-6 and n-3 polyunsaturated fatty acids generated by lipoxygenases are equally metabolized by the peroxide scavenging capabilities of the tissue.