Background & Aims: Cyclooxygenase (COX)-2 is up-regulated in most colonic cancers and in inflammatory bowel disease in which tumor necrosis factor (TNF)-α is believed to play a central role. There has been recent speculation on the activation of phosphatidylinositol 3-kinase (PI 3-kinase) by TNF-α and its role in the regulation of genes controlled by NF-κB. We investigated the regulatory role of PI 3-kinase on COX-2 expression in colonic epithelial cells. Methods: In HT-29 and Caco-2 colonic epithelial cells, COX-2 expression was induced by either TNF-α or interleukin (IL)-1α as observed by Northern and Western analyses. COX-2 activity was assessed by measuring prostaglandin E2 (PGE2) production by enzyme-linked immunosorbent assay. NF-κB binding activity was assessed by electrophoretic mobility shift assay. PI 3-kinase activity was measured by quantifying the accumulation of PI 3-kinase-dependent D-3 lipid products by high-performance liquid chromatography. Results: The PI 3-kinase inhibitor wortmannin up-regulated induced COX-2 expression in a concentration-dependent manner in both HT-29 and Caco-2 cells. An alternative PI 3-kinase inhibitor, LY294002, caused upregulation of induced COX-2 messenger RNA (mRNA) in HT-29 cells at concentrations of ≤1 μmol/L. IL-4 and IL-13, which are known to activate PI 3-kinase, downregulated HT-29 COX-2 mRNA, protein, and PGE2 production. NF-κB binding activity was unaltered by PI 3-kinase inhibition in HT-29 cells, in which TNF-α was shown to activate PI 3-kinase directly. Conclusions: COX-2 is negatively regulated by PI 3-kinase; we propose that the inhibitory effect of IL-4 and IL-43 is mediated via a PI 3-kinase-dependent pathway. This mechanism does not appear to involve NF-κB because PI 3-kinase inhibition did not alter NF-κB binding activity. TNF-α can activate PI 3-kinase directly in addition to inducing COX-2. Abbreviations: COX, cyclooxygenase; ELISA, enzyme-linked immunosorbent assay; HPLC, high-performance liquid chromatography; IBD, inflammatory bowel disease; IL, interleukin; iNOS, inducible nitric oxide synthase; NF-κB, nuclear factor-κB; NSAID, nonsteroidal anti-inflammatory drug; PGE2, prostaglandin E2; PGJ2, 15-deoxy δ12,14 prostaglandin J2; PI 3-kinase, phosphatidylinositol 3-kinase; PΚB, protein kinase B; Ptdlns, phosphatidylinositol; MAP, kinase mitogen-activated protein kinase; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; Th2, helper 2; TNF, tumor necrosis factor.