We have previously shown that PGE₂ and PGI₂ induce recovery of transepithelial resistance (TER) in ischemia-injured porcine ileal mucosa, associated with initial increases in Cl⁻secretion. We believe that the latter generates an osmotic gradient that stimulates resealing of tight junctions. Because of evidence implicating phosphatidylinositol 3-kinase (PI3K) in regulating tight junction assembly, we postulated that this signaling pathway is involved in PG-induced mucosal recovery. Porcine ileum was subjected to 45 min of ischemia, after which TER was monitored for a 180-min recovery period. Endogenous PG production was inhibited with indomethacin (5 μM). PGE₂ (1 μM) and PGI₂(1 μM) stimulated recovery of TER, which was inhibited by serosal application of the osmotic agent urea (300 mosmol/kgH₂O). The PI3K inhibitor wortmannin (10 nM) blocked recovery of TER in response to PGs or mucosal urea. Immunofluorescence imaging of recovering epithelium revealed that PGs restored occludin and zonula occludens-1 distribution to interepithelial junctions, and this pattern was disrupted by pretreatment with wortmannin. These experiments suggest that PGs stimulate recovery of paracellular resistance via a mechanism involving transepithelial osmotic gradients and PI3K-dependent restoration of tight junction protein distribution.