1. We evaluated the induction of endothelial barrier functions by the type 1 astrocyte-derived supernatant in culture using horseradish peroxidase (HRP) as a parameter. 2. A gel filtration fraction (molecular weight 16-20 kDa) of type 1 astrocyte-derived supernatant reduced plasmalemmal vesicles and induced tight junctions in endothelial cells. 3. In addition, this fraction promoted the uptake of [3H]-glucosamine by endothelial cells. This cell activity disappeared after heating at 95 degrees C for 10 min and was sensitive to trypsin. 4. These results suggest that a type 1 astrocyte-derived protein factor(s) promotes proteoglycan synthesis with a resultant increase in charge selectivity in endothelial cells and plays an important role in the induction of blood-brain barrier functions.