Oxidants such as monochloramine (NH₂Cl) decrease epithelial barrier function by disrupting perijunctional actin and possibly affecting the distribution of tight junctional proteins. These effects can, in theory, disturb cell polarization and affect critical membrane proteins by compromising molecular fence function of the tight junctions. To examine these possibilities, we investigated the actions of NH₂Cl on the distribution, function, and integrity of barrier-associated membrane, cytoskeletal, and adaptor proteins in human colonic Caco2 epithelial monolayers. NH₂Cl causes a time-dependent decrease in both detergent-insoluble and -soluble ZO-1 abundance, more rapidly in the former. Decreases in occludin levels in the detergent-insoluble fraction were observed soon after the fall of ZO-1 levels. The actin depolymerizer, cytochalasin D, resulted in a decreased transepithelial resistance (TER) more quickly than NH₂Cl, but caused a more modest and slower reduction in ZO-1 levels and in occludin re-distribution. No changes in the cellular distribution of claudin 1 or 5 or ZO-2 were observed after NH₂Cl. However, in subsequent studies, the immunofluorescent cellular staining pattern of all these proteins was altered by NH₂Cl. The actin stabilizing agent, phalloidin, did not prevent NH₂Cl-induced decreases in TER or increases of apical to basolateral flux of the paracellular permeability marker mannitol. However, it partially blocked changes in ZO-1 and occludin distribution. Tight junctional fence function was also compromised by NH₂Cl, observed as a redistribution of the alpha subunit of the basolateral Na⁺-K⁺-ATPase to the apical membrane, an effect not found with the apical membrane protein NHE3. In conclusion, oxidants not only disrupt perijunctional actin, but also cause redistribution of tight junctional proteins resulting in compromised intestinal epithelial barrier and fence function. These effects are likely to contribute to the development of malabsorption and dysfunction associated with mucosal inflammation of the digestive tract.