We have previously reported that the cytosolic phospholipase A₂ (cPLA₂) pathway is involved in ventilator-induced lung injury (VILI) produced by high peak inflation pressures (PIP) (Yoshikawa S, J. Appl. Physiol. 98:1264-71, 2005), but the relative contributions of the various downstream products of cPLA₂ on the acute permeability response was not determined. Therefore, we investigated the role of cPLA₂ and the downstream products of arachidonic acid metabolism on the high PIP ventilation induced increase in vascular permeability. We perfused isolated mouse lungs and measured the capillary filtration coefficient (Kfc) after 30-min periods of ventilation with PIP of 9, 25, and 35 cmH₂O. In high PIP ventilated lungs, Kfc increased significantly by 2.7-fold after ventilation with 35 cmH₂O PIP compared to paired baseline values and low PIP ventilated lungs. Also, an increased phosphorylation of lung cPLA₂ suggested enzyme activation after high PIP ventilation. However, treatment with 40 mg/kg arachidonyl trifluoromethyl ketone, an inhibitor of cPLA₂, or a combination of 30 µM ibuprofen, a cyclooxygenase inhibitor, 100 µM nordihydroguaiaretic acid, a lipoxygenase inhibitor, and 10 µM 17-octadecynoic acid, a cytochrome P450 epoxygenase inhibitor, prevented the high PIP induced increase in Kfc. Combinations of only one or two of the inhibitors of cyclooxygenase, lipoxygenase or P450 epoxygenase did not prevent significant increases in Kfc even though bronchoalveolar lavage levels of the cyclooxygenase or lipoxygenase products were significantly reduced. These results suggest that multiple mediators from each pathway contribute to the acute ventilator-induced permeability increase in isolated mouse lungs by mutual potentiation.