Epithelial cells undergo a form of apoptosis termed anoikis when they lose extracellular attachments. We evaluated the role of transcription factor NF-kappa B in the regulation of anoikis susceptibility of intestinal epithelial cells. Culture of rat intestinal epithelial cells in suspension induced NF-kappa B activation, which blocked the anoikis of those cells, as assessed by internucleosomal DNA fragmentation and caspase-3 cleavage. Activation of NF-kappa B after the loss of extracellular attachments required focal adhesion kinase tyrosine 397 phosphorylation. This triggered a signaling cascade through phosphatidylinositol 3-kinase and AKT, to induce DNA binding of the RelA/p65 NF-kappa B polypeptide. NF-kappa B activated in this manner induced the up-regulated expression of a distinct program of genes that included osteoprotegerin, BCL-2, and IAP-1 ( inhibitor of apoptosis protein-1). Chromatin immunoprecipitation experiments revealed that NF-kappa B directly regulated the promoters of these 3 genes. Knock-down of the expression of osteoprotegerin, BCL-2, or inhibitor of apoptosis protein-1 by RNA interference showed that these factors inhibit anoikis, and genetic reconstitution of their expression alone or in combination restored normal levels of anoikis to NF-kappa B-inactive intestinal epithelial cells. Together, these findings have identified the molecular components of a previously unrecognized antianoikis pathway in intestinal epithelial cells.