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Epidermal homogenates of hairless mice contained a large amount of lipid material showing a single peak on reverse phase (RP)-high pressure liquid chromatography (HPLC) with a maximum absorbance of 234 nm, which was different in retention time from arachidonate metabolites such as 12-HETE, 15-HETE and 5-HETE. The production of this material was dependent on exogenous linoleate concentration, protein concentration and buffer pH. This material was identified to be a mixture of 13-HODE and 9-NODE on the basis of UV absorbance spectra, retention times on RP-HPLC, straight phase (SP)-HPLC, gas chromatography-mass spectrometry (GC/MS) and mass spectra obtained. As assessed by SP-HPLC after saponification, these two HODEs were found to exist primarily in esterified forms in the skin. Neither substance was produced following heat denaturation of the homogenate. These results indicate that 13-and 9-HODEs are produced from linoleate enzymatically at physiologically relevant levels in the epidermis of hairless mice. Thus, it is possible that these two materials may play some important role in pathophysiology of the skin.