Rat alveolar macrophages were prelabeled with 3H-arachidonic acid (3H-AA) and exposed to air or O3 (0.1-1.0 ppm) in vitro for 2 h. Alveolar macrophages released 3.6-fold more tritium at the 1.0 ppm exposure concentration compared with air-exposed macrophages. A significantly increased production of several 3H-AA metabolites, including 6-keto-PGF1 alpha, thromboxane B2, 12-hydroxy-5,8,10-heptadecatrienoic acid, prostaglandins E2 and D2, leukotrienes B4 and D4, and 15-hydroxyeicosatetraenoic acid was formed by macrophages exposed to 1.0 ppm O3 compared with air-exposed macrophages as determined by high performance liquid chromatography (HPLC) analysis. O3 exposure did not alter macrophage 3H-AA metabolism in response to calcium ionophore A23187. The largest tritiated peak observed in the HPLC chromatograms of O3-exposed cells was a polar complex of products that contained various phospholipids and neutral lipids (including diacylglycerol) and possibly degradation products of 3H-AA and some of its metabolites. These changes in macrophage arachidonic acid metabolism may play an important role in the lung response to O3 exposure in vivo.