The predominating prostaglandins of human seminal fluid are 19R-hydroxyprostaglandins E1 and E2, conceivably formed sequentially by prostaglandin H (PGH) synthase-2, PGH 19-hydroxylase (CYP4F8), and microsomal PGE synthase-1 of seminal vesicles. Our aim was to study this enzyme system. Quantification by real-time PCR suggested that the transcripts of PGH synthase-2, CYP4F8, and microsomal PGE synthase-1 were abundant and correlated in seminal vesicles of seven patients (p < 0.05). The three enzymes were detected in seminal vesicles by Western blot analysis, and immunohistological analysis confirmed the localization to the epithelia of seminal vesicles and distal vas deferens. Immunofluorescence analysis showed co-localization of the three enzymes in epithelial cells of seminal vesicles and vas deferens. 19-Hydroxy-PGE compounds were detected by mass spectrometry in the mucosa of distal vas deferens. Recombinant CYP4F8 catalyzes n-2 hydroxylation of PGH1 and PGH2 and n-3 hydroxylation of arachidonic acid. Arachidonic acid was oxidized to 18-hydroxyarachidonic acid and to PGE2 and by microsomes of seminal vesicles in the presence of NADPH and GSH, and to relatively small amounts of 19-hydroxy-PGE2. We conclude that PGH synthase-2, CYP4F8, and PGE synthase-1 likely forms 19-hydroxy-PGE compounds in seminal vesicles and vas deferens, but the catalytic properties of CYP4F8 suggest additional biological functions. Recombinant CYP4F8 was also found to catalyze n-2 hydroxylation of PGI2 and carbaprostacyclin (Km to approximately 40 microM), and n-2 and n-3 hydroxylation of carbocyclic TXA2.