The blood-brain barrier (BBB) is involved in many normal regulatory mechanisms as well as in pathologic conditions of the central nervous system. Previous studies examining the development and function of the BBB in vitro have primarily utilized cell lines or cultured tissues from non-human sources. In contrast, this study used a coculture system of human fetal astrocytes and autologous endothelial cells. Astrocytes and endothelial cells (EC) were isolated and cultured on the opposite sides of a synthetic permeable membrane. The cocultures were characterized by electron and light microscopy for morphology and by immunocytochemistry for cell-type specific markers. Using these coculture conditions, astrocytes displayed characteristic morphology and expressed glial fibrillary acidic protein. When cocultured with astrocytes, endothelial cells retained factor VIII expression and expressed the BBB-specific proteins, brain-type glucose transporter (GLUT-1) and gamma-glutamyl transpeptidase. This expression was dependent on EC being in close apposition to or in direct contact with astrocytes. The model presented in this study may permit further examination of the role of the BBB in both normal human neurodevelopment and neuropathologic conditions.