The metabolism of [1-14C]arachidonic acid by microsomal and purified prostaglandin (PG) H synthase was investigated. HPLC analysis confirmed that arachidonic acid (20:4) was extensively converted into prostaglandin G2 (PGG2) and/or prostaglandin H2 (PGH2) but several minor labelled products were formed in addition. Their formation, mediated by PGH synthase was established by inhibition with aspirin and indomethacin [Hecker, M., Hatzelmann, A. & Ullrich, V. (1987) Biochem. Pharmacol. 36, 851-855]. Upon comparison with authentic reference material these unknown PGH synthase metabolites were identified with respect to chromatographic properties, ultraviolet spectroscopy and mass spectrometry as 11 (R)-hydroperoxy-5Z,8Z,12E,14Z-eicosatetraenoic acid (11-OOH-20:4), 12(S)-hydroperoxy-5Z,8E,10E-heptadecatrienoic acid (OOH-17:3), 12(S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid (OH-17:3), 15(RS)-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoi c acid (15-OOH-20:4), 15(RS)-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-OH-20:4), 13-hydroxy-5Z,14Z-prostaglandin H2, 15(S)-hydroxy-8-iso-5Z,13E-prostaglandin H2 and 15-oxo-prostaglandin H2. Unlike PGG2 and PGH2, 8-iso-PGH2, 13-hydroxy-PGH2 and 15-oxo-PGH2 failed to induce aggregation of washed human platelets and to form thromboxane upon incubation with homogeneous human platelet thromboxane synthase. In contrast to the formation of OOH-17:3, 15-oxo-PGH2 and OH-17:3 which can be attributed to the heme-catalyzed decomposition of PGG2 and PGH2, 11-OOH-20:4,15-(O)OH-20:4-,8 iso-PGH2 and 13-hydroxy-PGH2 represent potential side products of arachidonic acid conversion into PG endoperoxides. Their formation allows to conclude on PGH synthase mechanism and its intermediates for which an extended reaction scheme is proposed.