Although prostaglandins (PGs) have been detected in the middle ear fluids, the exact identification of PGs, and other arachidonic acid (AA) metabolites has not been made. The AA metabolites were identified in experimentally induced serous otitis media and purulent otitis media in chinchillas using a radiochromatography method. The relative abundance of each metabolite synthesized was also determined. Two major AA metabolites, 6-Keto-PGF1α and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), were positively identified by gas chromatography-mass spectrometry. The AA metabolites in Eustachian tube obstructed middle ear mucosa (MEM) were predominantly 6-keto-PGF1α, followed by PGD2, 15-HETE, hydroxyheptadecatrienoic acid (HHT), PGF2α and PGE2. In infected MEM, the predominant product was 15-HETE followed by 6-keto-PGF1α, PGD2, HHT, PGF2α, and PGE2. The possible role of PGs and other AA metabolites in the pathogenesis of otitis media will be discussed.