The objective was to examine the NADPH-dependent oxygenation of prostaglandin H₂ (PGH₂) and three PGH₂ analogues, 9,11-diazo-15-deoxy-PGH₂ (U51605), 9,11-epoxymethano-PGH₂ (U44069), and 11,9-epoxymethano-PGH₂ (U46619), by cytochromes P450, and to characterize the metabolites by mass spectrometry. CYP2C19, CYP4A11, CYP4F8, and liver and renal cortical microsomes oxidized the ω-side chain of U44069, U46619, and U51605, whereas only CYP4F8 oxidized the ω-side chain of PGH₂. PGH₂ was transformed to four stereoisomers of 5-hydroxy-PGI₁ by recombinant cytochromes P450. CYP4F8 formed the 5-hydroxy-PGI₁ isomers in small amounts compared to the 19-hydroxy metabolites of PGH₂. Isomers of 5-hydroxy-PGI₁ and 6-keto-PGF_1α were detectable when PGH₂ decomposed in the presence of hemin, hemoglobin, or heat-inactivated microsomes. 5-Hydroxy-PGI₁ is likely formed from PGH₂ 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 PGH₂, but the absolute configuration of the 19-hydroxy group is unknown, whereas human seminal fluid contains (19R)-hydroxy-PGE₂. 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 ω-side chain hydroxylation of PGH₂ analogues may be catalyzed by many different cytochromes P450, but only CYP4F8 oxidizes the ω-side chain of PGH₂ efficiently.