To study the blood-brain barrier in vitro pure cerebral endothelial cell cultures, without contaminating cells have to be obtained. Most other cell types besides endothelial cells can be pericytes, a few astrocytes, some smooth muscle cells, fibroblasts and meningeal cells. Careful removal of large vessels and meninges during the dissection and the optimal duration of enzymic digestions can reduce the ratio of contaminant cells. In order to further increase the purity of the culture endothelial cells can be subcloned, however, this is not useful for cells of every species. An alternative choice in cultures from rat is to perform a selective cytolysis by complement and monoclonal anti-Thy 1.1 antibody to eliminate pericytes and astrocytes. The presence of growth factors and the type of serum are also important for successful endothelial cell cultures. With the combination of the cytolysis of contaminating cells and the use of plasma-derived serum, the culturing of pure primary cerebral endothelial cells was successful.