Diseases of gut inflammation such as neonatal necrotizing enterocolitis (NEC) result after an injury to the mucosal lining of the intestine, leading to translocation of bacteria and endotoxin (lipopolysaccharide). Intestinal mucosal defects are repaired by the process of intestinal restitution, during which enterocytes migrate from healthy areas to sites of injury. In an animal model of NEC, we determined that intestinal restitution was significantly impaired compared with control animals. We therefore sought to determine the mechanisms governing enterocyte migration under basal conditions and after an endotoxin challenge. Here we show that the cytoskeletal reorganization and stress fiber formation required for migration in IEC-6 enterocytes requires RhoA. Enterocytes were found to express the endotoxin receptor Toll-like receptor 4, which served to bind and internalize lipopolysaccharide. Strikingly, endotoxin treatment significantly inhibited intestinal restitution, as measured by impaired IEC-6 cell migration across a scraped wound. Lipopolysaccharide was found to increase RhoA activity in a phosphatidylinositol 3-kinasedependent manner, leading to an increase in phosphorylation of focal adhesion kinase and an enhanced number of focal adhesions. Importantly, endotoxin caused a progressive, RhoA-dependent increase in cell matrix tension/contractility, which correlated with the observed impairment in enterocyte migration. We therefore conclude that endotoxin inhibits enterocyte migration through a RhoA-dependent increase in focal adhesions and enhanced cell adhesiveness, which may participate in the impaired restitution observed in experimental NEC.Necrotizing enterocolitis (NEC) 1 is the leading cause of death from gastrointestinal disease in neonates and is second to respiratory disease as the overall leading cause of morbidity and mortality in this population (1-3). Clinical manifestations of NEC include abdominal distention, feeding intolerance, and systemic sepsis, which result from the destruction of the intestinal barrier (4, 5). Mucosal breakdown may occur as a result of a perinatal insult, such as hypoxia, which allows bacteria and bacterial by-products to breach the normally impermeable mucosal barrier and initiate a local and systemic inflammatory response (4,6,7,9). This causes the release of proinflammatory cytokines, including tumor necrosis factor, platelet-activating factor, nitric oxide, and endotoxin, which lead to further epithelial injury (10 -13). Following mucosal damage, healing occurs initially through the process of epithelial restitution, in which healthy enterocytes adjacent to the sites of injury migrate toward the denuded mucosa to bridge the defect (14 -18). We hypothesize that the intestinal barrier defect in NEC does not result from epithelial injury alone but also from impaired restitution. This paper focuses specifically on the process of enterocyte migration, which is the sine qua non of epithelial restitution and mucosal healing.Although a variety of cytokines may be importan...