Cerebral endothelial cells form the selective permeability barrier between brain and blood by virtue of their impermeable tight junctions and the presence of specific carrier systems. These specialized properties of brain capillaries are reflected in the presence of proteins that are not found in other capillaries of the body. gamma-Glutamyltranspeptidase (GGT) has been widely used as a marker for brain capillaries and differentiated properties of brain endothelial cells. By using histochemical and biochemical methods we have investigated the expression of GGT in isolated capillaries, cultured brain endothelial cells and pericytes, and cocultures of astrocytes and brain endothelial cells. It was surprising that the majority of GGT activity was associated with pericytes, but not endothelial cells, suggesting that GGT is a specific marker for brain pericytes. The remaining GGT activity that was associated with endothelial cells rapidly disappeared from cultured cells but was reinduced in cocultures with astrocytes. Our results emphasize the need for pure endothelial cells for the investigation of blood-brain barrier characteristics.