Ozone exposure,in vitro,has been shown to activate phospholipases A₂(PLA₂), C (PLC), and D (PLD) in airway epithelial cells. However, because of its high reactivity, ozone cannot penetrate far into the air/lung tissue interface. It has been proposed that ozone reacts with unsaturated fatty acids (UFA) in the epithelial lining fluid (ELF) and cell membranes to generate a cascade of lipid ozonation products (LOP) that mediate ozone-induced toxicity. To test this hypothesis, we exposed cultured human bronchial epithelial cells (BEAS-2B) to LOP (1–100 μM) produced from the ozonation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and measured the activity of PLA₂, PLC, and PLD. The PLA₂isoform responsible for arachidonic acid release (AA) in stimulated cultures was also characterized. Activation of PLA₂, PLC, and PLD by three oxidants, hydrogen peroxide (H₂O₂),tert-butyl hydroperoxide (t-BOOH) and 2,2′-azobis(2-amidinopropane)dihydrochloride (AAPH) also was measured and compared to that of LOP. The derivatives of ozonized POPC at thesn-2 residue, 9-oxononanoyl (PC-ALD), 9-hydroxy-9-hydroperoxynonanoyl (PC-HHP), and 8-(-5-octyl-1,2,4-trioxolan-3-yl-) octanoyl (POPC-OZ) selectively activated PLA₂in a dose-dependent fashion. Cytosolic PLA₂(cPLA₂) measured in the cytosolic fraction of stimulated cell lysates was found to be the predominant isoform responsible for AA release. PLC activation was exclusively induced by the hydroxyhydroperoxide derivatives. PC-HHP and the 9-carbon hydroxyhydroperoxide (HHP-C9) increased PLC activity. PLD activity also was induced by LOP generated from POPC. Incubation of cultures with H₂O₂alone did not stimulate PLC; however, in the presence of the aldehyde, nonanal, a 62 ± 2% increase in PLC activity was found, suggesting that the increase in activity was due to the formation of the intermediate HHP-C9. t-BOOH, and AAPH also failed to induce PLA₂activation, but did activate PLC, under conditions of exposure identical to that of LOP. Only t-BOOH activated PLD. These results suggest that biologically relevant concentrations of LOP activate PLA₂, PLC, and PLD in the airway epithelial cell, a primary target to ozone exposure. The activation of these phospholipases may playa role in the development of lung inflammation during ozone exposure.