We have compared the ability of Müller cells and astrocytes to induce the formation of barrier properties in blood vessels. Müller cells cultured from the rabbit retina, and astrocytes and meningeal cells cultured from the rat cerebral cortex, were injected into the anterior chamber of the rat eye, where they formed aggregates on the iris. We have examined the barrier properties of the vessels in those aggregates and, for comparison, the barrier properties of vessels in the retina, ciliary processes and iris. Two tracers were perfused intravascularly to test barrier properties. The movement of Evans Blue was assessed by light microscopy, and the movement of horseradish peroxidase by light and electron microscopy. Our results indicate that Müller cells share the ability of astrocytes to induce the formation of barrier properties by vascular endothelial cells, and we suggest that Müller cells play a major role in the formation of barrier properties in retinal vessels.