Blood vessels in the central nervous system (CNS) are unique in forming the blood-brain barrier (BBB), which confers high electrical resistance and low permeability properties, thus protecting neural cells from potentially harmful blood components. Endothelial cells, which form the inner cellular lining of all blood vessels, play a critical role in this process by forming tight adhesive interactions between each other. To study the properties of primary brain endothelial cells (BECs), a number of different methods have been described. In this chapter, we present a relatively simple method that produces high numbers of primary mouse BECs that are highly pure (greater than 99 % CD31-positive). In addition, we also describe an immunocytochemical approach to demonstrate the endothelial purity of these cultures.