BACKGROUND and AIMS: The epithelial lining of the intestine serves as a barrier to lumenal bacteria and can be compromised by pathologic Fas-mediated epithelial apoptosis. Phosphatidylinositol (PI)3'-kinase signaling has been described to limit apoptosis in other systems. We hypothesized that PI3'-kinase-dependent pathways regulate Fas-mediated apoptosis and barrier function in intestinal epithelial cells (IEC). METHODS: IEC lines (HT-29, T84) were exposed to agonist anti-Fas Ab in the presence or absence of chemical inhibitors of PI3'-kinase (LY294002, wortmannin). Apoptosis, barrier function, changes in short-circuit current (change in Isc) and expression of adhesion molecules were assessed. RESULTS: Inhibition of PI3'-kinase strongly sensitized IEC to Fas-mediated apoptosis. Expression of constitutively-active Akt, a principal downstream effector of the PI3'-kinase pathway, protected against Fas-mediated apoptosis to an extent that was comparable to expression of a genetic caspase inhibitor, p35. PI3'-kinase inhibition sensitized to apoptosis by increasing and accelerating Fas-mediated caspase activation. Inhibition of PI3'-kinase combined with cross-linking Fas was associated with increased permeability to molecules that were <400D but not those >3000D. Inhibition of PI3'-kinase resulted in chloride secretion that was augmented by cross-linking Fas. Confocal analyses revealed polymerization of actin and maintenance of EpCAM-mediated interactions in monolayers exposed to anti-Fas Ab in the context of PI3'-kinase inhibition. CONCLUSIONS: PI3'-kinase-dependent pathways, especially Akt, protect IEC against Fas-mediated apoptosis. Inhibition of PI3'-kinase in the context of Fas signaling results in increased chloride secretion and barrier dysfunction. These findings suggest that agonists of PI3'-kinase such as growth factors may have a dual effect on intestinal inflammation, by protecting epithelial cells against immune-mediated apoptosis and limiting chloride secretory diarrhea.