Inflammation resulting from bacterial infection of the respiratory mucosal surface during pneumonia and cystic fibrosis contributes to pathology. A major consequence of the inflammatory response is recruitment of polymorphonuclear cells (PMNs) to the infected site. To reach the airway, PMNs must travel through several cellular and extra-cellular barriers, via the actions of multiple cytokines, chemokines, and adhesion molecules. Using a model of polarized lung epithelial cells (A549 or Calu-3) grown on Transwell filters and human PMNs, we have shown that Pseudomonas aeruginosa induce PMN migration across lung epithelial barriers. The process is mediated by epithelial production of the eicosanoid hepoxilin A₃ (HXA₃) in response to Pseudomonas aeruginosa infection. HXA₃ is a PMN chemo-attractant metabolized from arachidonic acid (A.A.). Given that release of A.A. is believed to be the rate-limiting step in generating eicosanoids, we investigated whether Pseudomonas aeruginosa infection of lung epithelial cells resulted in an increase in free A.A.. Pseudomonas aeruginosa infection of A549 or Calu-3 monolayers resulted in a significant increase in 3H-A.A. released from pre-labeled lung epithelial cells. This was partially inhibited by PLA₂ inhibitors ONO-RS-082 and ACA as well as an inhibitor of diacylglycerol lipase. Both PLA₂ inhibitors dramatically reduced Pseudomonas aeruginosa induced PMN transmigration, whereas the diacylglycerol lipase inhibitor had no effect. In addition, we observed that Pseudomonas aeruginosa infection caused an increase in the phosphorylation of cytosolic PLA₂ (cPLA₂), suggesting a mechanism whereby Pseudomonas aeruginosa activates cPLA₂ generating free A.A. that may be converted to HXA₃, which is required for mediating PMN transmigration.