The objective was to examine the NADPH-dependent oxygenation of prostaglandin H(2) (PGH(2)) and three PGH(2) analogues, 9,11-diazo-15-deoxy-PGH(2) (U51605), 9,11-epoxymethano-PGH(2) (U44069), and 11,9-epoxymethano-PGH(2) (U46619), by cytochromes P450, and to characterize the metabolites by mass spectrometry. CYP2C19, CYP4A11, CYP4F8, and liver and renal cortical microsomes oxidized the omega-side chain of U44069, U46619, and U51605, whereas only CYP4F8 oxidized the omega-side chain of PGH(2). PGH(2) was transformed to four stereoisomers of 5-hydroxy-PGI(1) by recombinant cytochromes P450. CYP4F8 formed the 5-hydroxy-PGI(1) isomers in small amounts compared to the 19-hydroxy metabolites of PGH(2). Isomers of 5-hydroxy-PGI(1) and 6-keto-PGF(1 alpha) were detectable when PGH(2) decomposed in the presence of hemin, hemoglobin, or heat-inactivated microsomes. 5-Hydroxy-PGI(1) is likely formed from PGH(2) in a pseudo-enzymatic reaction involving homolytic scission of the endoperoxide and formation of an ether between C-9 and C-6 and a carbon-centered radical at C-5, which reacts with molecular oxygen. CYP4F8 catalyzes 19-hydroxylation of PGH(2), but the absolute configuration of the 19-hydroxy group is unknown, whereas human seminal fluid contains (19R)-hydroxy-PGE(2). CYP4F8 was found to metabolize U51605 to 90% of the (19R)-hydroxy metabolite, providing further evidence in favor of a role of CYP4F8 in biosynthesis of (19R)-hydroxy PGE in human seminal vesicles. We conclude that omega-side chain hydroxylation of PGH(2) analogues may be catalyzed by many different cytochromes P450, but only CYP4F8 oxidizes the omega-side chain of PGH(2) efficiently.