Abstract The biosynthesis of leukotrienes (LTs) and 5-hydroxy-6,8,-11,14(E,Z,Z,Z)-eicosatetraenoic acid (5-HETE) in perfused guinea pig lung was studied by reversed phase-high-performance liquid chromatography (RP-HPLC). Perfusion of lungs with a 20 μ solution of ionophore A23187 for 25 minutes induced a sustained release of 5S-hydroxy-6R-S-cysteinylglycyl-7,9,11,14-(E,E,Z,Z)eicosatetraenoic acid (LTD 4) and 5S,12R-dihydroxy-6,8,10,14(Z,E,E,Z)-eicosatetraenoic acid (LTB 4). The perfusion of 60 νM of arachidonic acid together with the ionophore only slightly increased the synthesis of LTD, and LTB, in comparison with the effect of the ionophore alone but markedly stimulated the release of 5-HETE that was not detectable under other experimental conditions. Immunologic challenge of the lung by continuous perfusion of ovalbumin to previously sensitized guinea pigs induced the release of 50 to 150 νM of LTB, and 15 to 50 pM of LTD 4 during a 25-minute period. Antigen was a less potent stimulus than the ionophore that caused the release of 500 to 900 pM of LTB, and 100 to 250 pM of LTD, during 25minutes of perfusion. None of the above-mentioned metabolites were detected when lungs were perfused with arachidonic acid alone, indicating that the synthesis of 5-lipoxygenase products required activation of the lung. In addition to 5-lipoxygenase products, the cyclooxygenase products 12-hydroxy-5,8,10-(Z,E,E)-heptadecatrienoic acid and 12-keto-5,8,10-(Z,E,E)heptadecatrienoic acid were analyzed by RP-HPLC in guinea pig lung perfusates. HPLC analysis provided quantitative data on the release of several arachidonic acid metabolites by lungs. These studies also pointed out the potential importance of lung-derived LTB, in anaphylactic responses.