Every blood vessel in the body is lined with a specialized layer of polarized cells known as endothelium. An essential function of the endothelial monolayer is the regulation of barrier integrity, which prevents the leakage of plasma and proteins out of the circulation while still permitting the flux of nutrients and immune cells to target tissues.In principle, permeability of the endothelial monolayer can reflect contributions from leaking between endothelial cells (paracellular leak) and through individual endothelial cells (transcellular leak, or transcytosis). It is widely accepted that paracellular leak predominates during inflammatory states such as sepsis and acute lung injury. Accordingly, by far the majority of research on endothelial permeability has focused on this route of endothelial permeability: the methods of study are relatively straight-forward and there is obvious relevance to human disease. In contrast, the contribution of transcytosis to overall endothelial permeability is relatively obscure, particularly in the setting of inflammation. This is largely due to technical difficulties in distinguishing transcellular permeability from intercellular gaps, particularly in a dynamic and quantifiable way. In addition, endothelial cells grown in culture appear to lose the ability to perform transcytosis as they are passaged. Much of the initial work on transcytosis used electron microscopy of animal tissues, an expensive and often a mostly descriptive endeavour. Transcytosis (at least in the apical to basal direction) is best described for the plasma protein albumin and is mediated by caveolae, small vesicles that bud off from the apical endothelial surface and release their cargo at the basal membrane. This process requires the protein caveolin-1 and the large GTPase dynamin; the latter is thought to mediate the scission of internalized caveolae from the apical plasmalemma.