[14C]Linoleic acid was incubated with human liver microsomes and NADPH and biosynthesis of allylic hydroxy fatty acids was investigated. 11-Hydroxy-9Z,12Z-octadecadienoic acid (11-HODE), 9-hydroxy-10E,12Z-octadecadienoic acid (9-HODE), 13-hydroxy-9Z,11E-octadecadienoic acid (13-HODE) and 14-hydroxy-9Z,12Z-octadecadienoic acid were identified. 9-HODE and 13-HODE were formed with stereoselectivity (80% R) provided that 11-HODE was prevented from decomposing to 9(R,S)-HODE and 13(R,S)-HODE by extractive isolation at pH 5-6. Human hepatic microsomes metabolized [14C]arachidonic acid to many products, including 13-HETE and small amounts of 15-HETE (> 90% R), 11-HETE (59% R) and 12-HETE (> 90% R). Hepatic microsomes of untreated rats metabolized [14C]linoleic acid to 11-HODE as a major product, but significant formation of 11-HODE by purified cytochrome P-450 (P450) (CYP1A1, CYP2B1, CYP2B4, CYP2E1, CYP3A6 and CYP4A1) in a reconstituted system could not be detected, indicating that 11-HODE might be formed by other and constitutive P450 isozymes.