Recombinant CYP4F8 and CYP4F12 metabolize prostaglandin H 2 (PGH 2 ) analogs by ω2- and ω3-hydroxylation and arachidonic acid (20:4n-6) by ω3-hydroxylation. CYP4F8 was found to catalyze epoxidation of docosahexaenoic acid (22:6n-3) and docosapentaenoic acid (22:5n-3) and ω3-hydroxylation of 22:5n-6. CYP4F12 oxidized 22:6n-3 and 22:5n-3 in the same way, but 22:5n-6 was a poor substrate. The products were identified by liquid chromatography–mass spectrometry. The missense mutation 374A>T of CYP4F8 (Tyr 125 Phe in substrate recognition site-1 (SRS-1)) occurs in low frequency. This variant oxidized two PGH 2 analogs, U-51605 and U-44069, in analogy with CYP4F8, but 20:4n-6 and 22:5n-6 were not oxidized. CYP4F enzymes with ω-hydroxylase activity contain a heme-binding Glu residue, whereas CYP4F8 (and CYP4F12) with ω2- and ω 3-hydroxylase activities has a Gly residue in this position of SRS-4. The mutant CYP4F8 Gly 328 Glu oxidized U-51605 and U-44069 as recombinant CYP4F8, but the hydroxylation of arachidonic acid was shifted from C-18 to C-19. Single amino acid substitutions in SRS-1 and SRS-4 of CYP4F8 may thus influence oxygenation of certain substrates. We conclude that CYP4F8 and CYP4F12 catalyze epoxidation of 22:6n-3 and 22:5n-3, and CYP4F8 ω3-hydroxylation of 22:5n-6.