Interleukins 4 and 13 can affect their target cells by activation of signal transducer and activator of transcription 6 (STAT 6) or phosphatidylinositol 3-kinase (PI3K). We examined the signal transduction events involved in IL-4 and IL-13 regulation of epithelial paracellular permeability using T84 cells, a model human colonic epithelium. T84 cells treated with IL-4 or IL-13 displayed virtually identical dose- and time-dependent STAT 6 activation as assessed by electrophoretic mobility shift assay (EMSA) and decreases in transepithelial resistance (TER). STAT 6 DNA binding activity was maximal in nuclear extracts 30 min after exposure to IL-4 or IL-13, and TER was maximally reduced by 24 h post-treatment. Pretreatment of epithelia with transcription factor decoys (phosphorothioated DNA oligonucleotides containing the STAT 6 binding site) dramatically reduced STAT 6 activation as detected by EMSA, but did not attenuate the TER reduction by IL-4 or IL-13. In contrast, although the PI3K inhibitors wortmannin and LY294002 did not affect IL-4 or IL-13 STAT 6 activation, they significantly inhibited the ability of either cytokine to lower TER. Thus, we provide evidence for PI3K as the major proximal signaling event in IL-4 and IL-13 regulation of TER and speculate that pharmacological targeting of enterocytic PI3K activity may represent a means to manipulate epithelial permeability.