Until recently, microvascular endothelial cells of the blood-brain barrier were considered a homogeneous population, whose properties were induced by astrocytes. To address the question of brain endothelial cell heterogeneity, we measured the activity of specific markers of endothelial cells of different origins, including two brain-derived endothelial cell lines grown on plastic or on extracellular matrix (ECM) components secreted either by a glial or a fibroblast cell line, with or without the addition of astrocyte-conditioned medium (ACM). ECM components and ACM conjointly, but differently in each endothelial cell line, increased the uptake of leucine and modulated the expression of the specific markers alkaline phosphatase (ALP) and gamma-glutamyl transpeptidase (GGTP) in both cerebral endothelial lines. ACM decreased the incorporation of thymidine in the pulmonary endothelial cell line, but not in a cerebral cell line. An induction of blood-brain barrier specific GGTP was never observed in endothelial cells of non-cerebral origin. These results demonstrate that subpopulations of cerebral endothelial cells exist, that astrocyte-derived soluble factors and ECM components can modulate the expression and the function of markers of endothelial cells and that astrocyte-derived soluble factors have a selective growth inhibitory effect on endothelial cells of non-cerebral origin