The ubiquitous transcription factor NF-kappa B, which is activated in cells by diverse stimuli including phosphatidylinositol 3-kinase (PI3-kinase), is a critical factor for cell survival and growth. Inhibition of PI3-kinase enhances enterocyte-like differentiation of the human colon cancer cell line HT-29. The purpose of our study was to determine whether PI3-kinase alters NF-kappa B in HT-29 cells. Wortmannin, a specific PI3-kinase inhibitor, stimulated NP-kappa B binding activity in HT-29 cells by 4 h after treatment. Activation of NF-kappa B occurred without degradation of I kappa B alpha, a protein that sequesters NF-kappa B in the cytosol. In addition to increasing NF-kappa B binding, either wortmannin or cotransfection with a dominant negative mutant of the p85 regulatory subunit of PI3-kinase (Delta p85) induced NF-kappa B transactivation. Taken together, these results suggest that inhibition of PI3-kinase in HT-29 cells results in induction of NF-kappa B binding activity and transactivation which is independent of I kappa B alpha degradation,