γ-Glutamyl transpeptidase (γGTP) is an enzyme found in cerebral capillary endothelial cells, the presumed site of the blood-brain barrier, but not in endothelial cells lining blood vessels in other parts of the body. Using a line of mouse cerebral microvessel endothelial cells (ME-ly cells) and a sensitive colorimetric assay to measure γGTP levels we demonstrated that primary cultures of mouse astrocytes and a line of rat C₆ glioma cells released a soluble product(s) that induced the production of γGTP in cultured endothelial cells by 34% and 39%, respectively, over control levels. Cerebrovascular smooth muscle cells had no significant effect on γGTP levels in ME-ly cells, and the astrocyte product(s) had no effect on rabbit aortic endothelial cells. The induction of γGTP levels in ME-ly cells was apparent after one day of exposure to the astrocyte product(s) and increased in magnitude with increasing time of exposure of the ME-ly cells to the product(s). Removal of the product(s) from the ME-ly cells resulted in a return to control levels of γGTP in the ME-ly cells within 2 days. The presence of a protein synthesis inhibitor during incubation with the productd(s) blocked the induction of γGTP in ME-ly cells, and treatment of the product(s) with 200 U/ml TPCK-trypsin destroyed its inductive properties. These results show that glial cells release a protein into their medium which induces de novo protein synthesis of γGTP in cerebral microvessel endothelial cells, and indicate that the glial cells may induce the cerebral capillary endothelial cells to express differentiated properties which allow the endothelium to function as the blood-brain barrier.