We describe high-performance liquid chromatography (HPLC) methods for the enantiomeric resolution of hydroxy and hydroperoxy fatty acids/eicosanoids using a Chiralpak AD or AD-RH chiral stationary phase. These columns achieve baseline resolution of all six positional/conjugated isomers of the hydroxy as well as of the hydroperoxy derivatives of arachidonic acid in chromatographic runs of less than 20 min. Hydro(pero)xy derivatives of linoleic and linolenic acids can be resolved with similar efficiencies. The individual hydroperoxy isomers are best resolved using the reversed-phase Chiralpak AD-RH column. For the synthesis of milligram quantities of enantiomerically pure hydro(pero)xy arachidonic acids, a simple scheme is presented starting with the autoxidation of the fatty acid methyl ester in the presence of 10% alpha-tocopherol followed by chromatographic purification of the positional isomers using a combination of reversed- and straight-phase HPLC columns. Mild alkaline hydrolysis of the methyl ester derivatives affords the free acids suitable for biological testing. The Chiralpak AD column appears to be efficient for the chiral resolution of prostaglandins and isoprostanes although a comprehensive evaluation is yet to be reported. For chiral analysis of endogenous hydroxy eicosanoids the availability of novel microflow Chiralpak capillary columns (0.3 mm i.d.) will be of great advantage, because sample sizes of a few nanograms can be analyzed using simple UV detection.