The endothelium is a single-cell lining the blood vessels and represents an interface between blood and tissue. It acts as a selective permeability barrier, regulates coagulation and contributes to the behaviour of cells both in the circulation and in the vessel wall. Because of its location, one of the most important function of the endothelium is the regulation of the movement from the vascular to the extravascular space of water and solutes containing nutrients. Recent advances in our knowledge of the blood-brain barrier (BBB) have in part been made by studying the properties and function of cerebral endothelial cells (CECs) in vitro. After an era working with a fraction, enriched in cerebral microvessels by centrifugation, the next generation of in vitro BBB model systems was introduced, when the conditions for routinely culturing the endothelial cells were established. This review summarizes the results from this rapidly growing field. In addition to providing a better insight into the chemical composition of CECs, much has been learned from these studies about the characteristics of transport processes and cell-to-cell interactions during the last years. Astrocytes and neuronal elements contribute to the induction of BBB properties of CECs during ontogenesis and in tissue culture conditions. With the application of new technologies, the approach offers new means to investigation, applicable not only to biochemistry and physiology but also to the drug research, and may improve the transport of substances through the BBB. CECs grown on microporous cell culture inserts and co-cultured with astrocytes or treated by astrocyte-conditioned media proved to be excellent models for studying the direct effects of mediators and second messengers on the transendothelial permeability. The in vitro approach has been and should remain an excellent model of the BBB to help unravel the complex molecular interactions underlying and regulating the permeability of cerebral endothelium.