Docosahexaenoic acid, an n-3 essential fatty acid, was recently shown to be enzymically converted by platelets, basophils, and liver microsomes into metabolites containing conjugated dienes with allylic hydroxyl groups. To help identify these metabolites, standards were prepared by autooxidation of docosahexaenoic acid. After isolation by reverse phase and normal phase high performance chromatography (HPLC), ten hydroxy isomers of docosahexaenoic acid were identified by capillary gas-liquid chromatography, ultraviolet spectroscopy, and mass spectrometry. From these studies and reported elution orders for similar metabolites derived from linoleic, linolenic, and arachidonic acids, two basic HPLC elution patterns became apparent. Under reverse phase chromatography conditions, the distance of the trans-double bond from the carboxyl group was the critical parameter in determining the elution order. Under silicic acid chromatography conditions, the distance of the hydroxyl from the carbomethoxy group seemed to determine the elution order. The dramatic difference in selectivity between reverse and normal phase HPLC of the hydroxy acids provides critical information useful for identifying endogenous metabolites.