Metabolites of arachidonic acid are important regulators of biological function in a variety of mammalian tissues. We have demonstrated similar lipoxygenase enzyme activities in fish gills and mammalian lungs suggesting that their metabolites may have matching functions. Fish gills were investigated for their ability to generate dioxygenated metabolites of polyunsaturated fatty acids. Fatty acids, including arachidonic acid, were incubated with crude tissue homogenates and polar metabolites were extracted, derivatized and analyzed by high performance liquid chromatography, gas chromatography and mass spectrometry. The major dihydroxy metabolite of arachidonic acid was characterized as 8(L_R), 15(L_S)-dihydroxyeicosatetraenoic acid. This product was formed by the sequential action of the 15- and 12-lipoxygenases in the tissue. The formation of the dihydroxyeicosatetraenoic acid by crude tissue homogenates was significantly enhanced by the addition of 1 mM reduced glutathione. The metabolism of other polyunsaturated fatty acids, including eicosapentaenoic acid and docosahexaenoic acid, to dihydroxy acids was consistent with their relative ability to serve as substrates for the initial 15-lipoxygenase reaction.