An important function of the endothelium is to regulate the transport of liquid and solutes across the semi-permeable vascular endothelial barrier. Two cellular pathways controlling endothelial barrier function have been identified. The transcellular pathway transports plasma proteins of the size of albumin or greater via the process of transcytosis in vesicle carriers originating from cell surface caveolae. Specific signalling cues are able to induce the internalisation of caveolae and their movement to the basal side of the endothelium. Caveolin-1, the primary structural protein required for the formation of caveolae, is also important in regulating vesicle trafficking through the cell by controlling the activity and localisation of signalling molecules that mediate vesicle fission, endocytosis, fusion and finally exocytosis. An important function of the transcytotic pathways is to regulate the delivery of albumin and immunoglobulins, thereby controlling tissue oncotic pressure and host-defence. The paracellular pathway induced during inflammation is formed by gaps between endothelial cells at the level of adherens and tight junctional complexes. Paracellular permeability is increased by second messenger signalling pathways involving Ca2+ influx via activation of store-operated channels, protein kinase Calpha (PKCalpha), and Rho kinase that together participate in the stimulation of myosin light chain phosphorylation, actin-myosin contraction, and disruption of the junctions. In this review of the field, we discuss the current understanding of the signalling pathways regulating paracellular and transcellular endothelial permeability.