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Oxidative stress is thought to be a factor influencing many inflammatory responses, including arachidonic acid (AA) release. We have studied the effect of hydrogen peroxide on AA and prostaglandin E2 release, cytosolic phospholipase (cPLA2) steady-state mRNA, cPLA2 protein levels, cPLA2 enzyme activity, and cPLA2 phosphorylation in a human lung epithelial cell line: A549 cells. Hydrogen peroxide caused a dose-dependent increase of A23187-stimulated AA and prostaglandin E2 release, with a maximum effect at 1 h. This effect is associated with a maximum specific cPLA2 activity at 1 h, and with a significant increase in cPLA2 Serine 505 phosphorylation. All these effects were abolished, in a dose-related manner, by the epithelial growth factor receptor kinase inhibitor, AG 1478. To further investigate the pathway leading to the increase cPLA2 phosphorylation, we used cells transfected with a Ras dominant negative vector and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and p38 kinase inhibitors. Cells transfected with the Ras dominant negative vector exhibited diminished hydrogen peroxide-induced AA release and cPLA2 phosphorylation as compared with cells transfected with the Ras expression vector. Both MEK and p38 kinase inhibitors inhibited the hydrogen peroxide effect on AA release and specific cPLA2 activity. Finally, cells stably transfected with an antisense cPLA2 vector exhibited diminished A23187-stimulated AA release in response to hydrogen peroxide as compared with cells stably transfected with empty expression vector. Collectively, these data show that hydrogen peroxide increases cPLA2 activity through its phosphorylation utilizing an epithelial growth factor/Ras/extracellular signal-regulated kinase and p38 pathway.