Mechanical ventilation is the primary supportive treatment for infants and adults suffering from severe respiratory failure. Adverse mechanical ventilation (overdistension of the lung) triggers a proinflammatory response. Along with cytokines, inflammatory mediators such as bioactive lipids are involved in the regulation of the inflammatory response. The arachidonic acid pathway is a key source of bioactive lipid mediators, including prostanoids. Although ventilation has been shown to influence the production of prostanoids in the lung, the mechanotransduction pathways are unknown. Herein, we established that cyclic stretch of fetal lung epithelial cells, but not fibroblasts, can evoke an extremely sensitive, rapid alteration in eicosanoid metabolism through a cyclooxygenase (COX)-2 dependent mechanism. Cyclic stretch significantly increased PGI₂, PGF_2α, PGD₂, PGE₂, and thromboxane B₂ levels in the media of epithelial cells, but did not alter leukotriene B₄ or 12-hydroxyeicosatetraenoic acid levels. Inhibition of COX-2, but not COX-1, attenuated the cyclic stretch-induced PG increase in the media, suggesting that cyclic stretch primarily affected PG synthesis. Substrate (free arachidonic acid) availability for PG generation was increased because of a cyclic stretch-induced activation of cytosolic phospholipase A₂ (cPLA₂) via an influx of extracellular calcium and phosphorylation by mitogen-activated protein kinase, p44/42MAPK. The data are compatible with cPLA₂ and COX-2 being intimately involved in regulating the injury response to adverse mechanical ventilation.