(1) A distinct group of radial glia resides along the roofplate of the mesencephalon. Results of experiments, in which the neonatal tectum is manipulated surgically, point to the involvement of these glia in compartmentalizing retinotectal axons to one side of the midbrain. (2) Immunohistochemical studies document that the GAGs CS and KS are expressed along these midline glia during development: their expression occurs after the intertectal axons grow across the midline, but is coincident with the time of ingrowth of retinotectal axons, which fail to cross the midline. Together with results of in vitro experiments from other laboratories, these observations suggest that CS and KS are involved in the barrier function of the midline cells. (3) Preliminary data on biochemical characterization of PGs in developing tectum indicate that similar PG core proteins are found in the midline region as well as in the lateral tectum, whereas metabolic labeling shows a significantly higher uptake of radioactive sulfates along the midline. Thus differential glycosylation of proteins along the midline is likely, along with the possibility that it is the sugar chains which contribute to the barrier function of the raphe glia. Taken in the context of what we currently know about the biochemical heterogeneity of PGs, their developmental expression, and their functions in relation to the growth of axons from a variety of different neuronal cell types, it is clear that the analyses of interactions between PGs and growing axons must occur at several different levels, not the least of which involves a detailed understanding of the milieu in vivo within which these interactions take place.