Brain endothelial cells, a site of the blood–brain barrier in vivo, regulate a number of physiological and pathophysiological processes in the brain including transport of nutrients, export of critical toxins, transmigration of circulating leukocytes and formation of new blood vessels. In this report, we describe a simple and reproducible method to isolate pure (>99%), functionally active endothelial cells from small quantities of adult mouse brain tissue. In vitro, these cells express typical phenotypic markers of differentiated brain endothelium such as von Willebrand factor, multiple drug resistant protein and glucose transporter-1, demonstrate uptake of acetylated low-density lipoprotein, and possess morphological and ultrastructural characteristics of microvascular endothelium. They form tight junctions and capillary-like tubes when stimulated by growth factors in an in vitro angiogenesis assay. In response to tumor necrosis factor-α, isolated mouse brain endothelial cells (MBEC) express vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). The protocol described here provides an effective and reliable method to isolate pure cerebral endothelium from adult mouse brain that should offer a useful tool for studying the role of altered vascular biology in mice with genetically manipulated brain disorders.