8-Hydroxyoctadeca-9 Z,12 Z-dienoic acid (8-HODE) and 10-hydroxyoctadeca-8 E,12 Z-octadecadienoic acid (10-HODE) are produced by fungi, e.g., 8 R-HODE by Gaeumannomyces graminis (take-all of wheat) and Aspergillus nidulans, 10 S-HODE by Lentinula edodes, and 10 R-HODE by Epichloe typhina. Racemic [8- 2H]8-HODE and [10- 2H]10-HODE were prepared by oxidation of 8- and 10-HODE to keto fatty acids by Dess–Martin periodinane followed by reduction to hydroxy fatty acids with NaB 2H 4. The hydroxy fatty acids were analyzed by chiral phase high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) with 8 R-HODE and 10 S-HODE as standards. 8 R-HODE eluted after 8 S-HODE on silica with cellulose tribenzoate (Chiralcel OB-H), and 10 S-HODE eluted before 10 R-HODE on silica with an aromatic chiral selector (Reprosil Chiral-NR). 5 S,8 R-Dihydroxyoctadeca-9 Z,12 Z-dienoic acid (5 S,8 R-DiHODE) is formed from 18:2n-6 by A. nidulans and 8 R,11 S-dihydroxyoctadeca-9 Z,12 Z-dienoic acid (8 R,11 S-DiHODE) by Agaricus bisporus. 8 R-Hydroperoxylinoleic acid (8 R-HPODE) can be transformed to 5 S,8 R-DiHODE and 8 R,11-DiHODE by Aspergillus spp., and 8 R,13-dihydroxy-9 Z,11 E-dienoic acid (8 R,13-DiHODE) can also be detected. We prepared racemic [5,8- 2H 2]5,8- and [8,11- 2H 2]8,11-DiHODE by oxidation and reduction as above and 8 R,13 S- and 8 R,13 R-DiHODE by oxidation of 8 R-HODE by S and R lipoxygenases. The diastereoisomers were separated and identified by normal phase HPLC–MS/MS analysis. We used the methods for steric analysis of fungal oxylipins. Aspergillus spp. produced 8 R-HODE (>95% R), 10 R-HODE (>70% R), and 5 S,8 R- and 8 R,11 S-DiHODE with high stereoselectivity (>95%), whereas 8 R,13-DiHODE was likely formed by nonenzymatic hydrolysis of 8 R,11 S-DiHODE.