Small intestinal epithelial cells migrate from the base of the crypt to the villus from where they are shed. A vital function of the small intestinal epithelium is to form a barrier against the luminal contents of the gut. Little is known how this is achieved in vivo. This question is of increasing relevance as data is accumulating that a defective intestinal barrier function plays a key role in the pathogenesis of inflammatory bowel disease. Experiments using confocal and multiphoton microscopy have demonstrated that discontinuities or “gaps” are left in the epithelium after epithelial cells are shed. Initial experiments showed that these gaps did not contain nuclei or cytoplasmic proteins and had the same diameter as an epithelial cell of 10 µm. Confocal microscopy was an essential technique in these initial experiments as it enabled three-dimensional reconstruction that showed that these gaps were artifacts of optical sectioning. Confocal endomicroscopy has demonstrated these gaps are present in human intestinal epithelium and are likely, in most cases, to be a space in the epithelial monolayer that results from shedding cells. Epithelial gaps can be distinguished from goblet cells as goblet cells possess a nucleus, have a characteristic appearance at confocal endomicroscopy and crucially are not seen in Math1 -/- mice which lack secretory epithelial cell lineages, including goblet cells in the distal small intestine. More recent work has shown that during the shedding of intestinal epithelial cells there is a profound distribution of ZO-1, occludin and actin from the tight junction to around the basolateral wall of the shedding cell forming a “funnel” which in most cases preserves barrier function. Treatment with TNF increases the rate of cell shedding. Apoptosis is likely to initiate cell shedding since it is inhibited by the caspase inhibitors ZVAD and Q-VD-OPh (50µM). After TNF barrier function at the site of cell shedding is lost in approximately 20% of cases. This loss of localized barrier function can be detected by both influx of luminal dyes or efflux of intravenous dyes such as Alexa dextrans through the epithelial gaps. In particular loss of barrier function at sites of cell shedding can be detected by confocal endomicroscopy in humans using intravenous fluorescein. No other technique is able to detect these abnormalities. Together, these studies show that the sites of epithelial cell shedding should be regarded along with the apical membrane of epithelial cells and the tight junction as a component of the intestinal permeability barrier. The increase in cell shedding and loss of barrier function following TNF may be an important part of the pathogenesis of inflammatory conditions of the intestine such as inflammatory bowel disease.