The specific structure of the blood-brain barrier (BBB) is based on the partnership of brain endothelial cells and astrocytes. In the last decade, cocultures of these two cell types have been developed as in vitro models. However, these studies did not allow close contacts between both cell types. We report here a syngenic coculture model using rat endothelial cells on one side of a polyethylene terephtalate filter and rat astrocytes on the other. Endothelial cells retain their typical morphology and are factor VIII and OX 26 positive. We optimized the diameter of the membrane pores to establish very close contacts between the cells through the membrane pores without mixing the two cell types. Transmission electron microscopy showed evidence of tight junction formation between the endothelial cells and few pinocytic vesicles. The cocultures reached high electrical resistances up to 1000 Omegacm(2) showing their ability to limit the passage of ions. A 15-fold increase in gamma-glutamyl transpeptidase activity was measured in the endothelial cells in coculture compared to endothelial cell monoculture. Our syngenic coculture represents a useful in vitro model of the rat BBB that may prove to be valuable for studying the passage of substances across the barrier as well as other aspects of the BBB function.