A highly reproducible in vitro model of intestinal epithelial injury in guinea pig ileum was used to study the structural and functional events that accompany rapid epithelial repair. This model is characterized by denudation of the villus tip followed by rapid restitution of the epithelial barrier. Using standard electrophysiological and quantitative morphometric techniques, we found that immediately after injury the number of cells lost exceeded the number of empty cell positions on the denuded basement membrane by 100%. Concurrently, cytoplasmic processes of subepithelial myofibroblasts contained condensations of microfilaments that were not apparent in controls. Additionally, villus height was diminished immediately after injury, and progressively decreased during the restitution period. In tissues that were depleted of ATP using the uncoupler dinitrophenol and in tissues functionally denervated by tetrodotoxin, villus shortening after injury was significantly reduced. Denervation also retarded functionally and structurally defined reestablishment of epithelial barrier function. These data suggest that intestinal epithelial repair is aided by energy-dependent, neurally mediated villus contraction. We speculate that the subepithelial network of myofibroblasts is responsible for this process, which effectively minimizes the denuded surface area to be reepithelialized.