Abstract [ 14C] Linolenic acid (18:ω3) and [ 14C] linoleic acid (18:2ω6) were incubated with hepatic microsomes of the rabbit in the presence of NADPH (1 Mm) for 15 min at 37°C. The products were extracted and purified by high performance liquid chromatography. The major metabolites of linolenic and linoleic acid were identified by capillary gas chromatography mass spectrometry as 15,16-dihydroxy-9,12-octadecadienoic acid, 12,13-dihydroxy-9,15-octadecadienoic acid and 9,10-dihydroxy-12,15-octadecadienoic acid and as 12,13-dihydroxy-9-octadecaenoic acid and 9,10-dihydroxy-12-octadecaenoic acid, respectively. The results were confirmed by comparison with mass spectra of the authentic compounds. These metabolites are presumably formed by cytochrome P-450 catalyzed epoxidation of the ω3, ω6 and ω9 double bonds, followed by enzymatic hydrolysis to 1,2-diols. The ratio of ω3, ω6 and ω9 oxygenated metabolites of linolenic acid was approximately 2:1:1 and the ratio of the ω6 and ω9 metabolites of linoleic acid was 2:1, indicating that the double bond closest the ω end is most easily oxygenated.