Radial glial cells and their processes play critical roles in organizing the spatial arrangement of the nervous system in the embryonic brain. It has been thought that following completion of their roles in the embryo, most of the radial glial processes disappear before or shortly after birth. Here we use R2D5, a monoclonal antibody to a soluble cytosolic protein, to demonstrate that a specific system of midsagittal radial glial cells persists in postnatal and adult brain. In the brainstem of postnatally developing and adult rabbits and cats, the R2D5-positive processes of radial glial cells were observed to be arranged in a precisely parallel array at the midsagittal seam. These radial glial processes formed a continuous palisade separating the right and left brainstem. In early postnatal animals, R2D5-positive radial processes were found to reach the pial surface and to cover the entire midsagittal seam of the brainstem. These processes embraced dendrites and somata of neurons in almost all of the midsagittal nuclei, including the raphe nuclei, suggesting that the radial glial cells may interact with the midsagittal groups of neurons. In addition, the palisade of R2D5-positive radial processes formed loose openings for crossing axonal bundles at the midline decussations of fiber tracts. In more mature brains, somata of R2D5-positive radial glial cells that had migrated ventrally were observed within the palisades, and in adult cats, most of the R2D5-positive radial processes were found to have retracted from the ventral parts of the midsagittal seam. The spatial arrangement of R2D5-positive processes suggests that they may have persistent functional roles as an interface between ventricular humoral signals and midsagittal groups of neurons in the postnatally developing brainstem and in the adult brainstem. The structure of the midline glial system suggests also that it plays a role in organizing the spatial arrangement of decussating axons during development.Radial glial cells are process-bearing ependymal cells lining the surface of brain ventricles and the central canal of the spinal cord. These are the first class of glial cells to appear during embryonic development and they are thought to play critical roles in organizing the spatial arrangement of the nervous system (1, 2). In lower vertebrates, for example, radial processes of ependymal cells have been reported to form channels for growing axons during both regeneration and embryonic development and thus have been suggested to display on their surfaces trace pathways that the axons follow toward their destination (3, 4). In the mammalian embryo, ependymal cells project their processes (radial glial fibers) to almost all regions of the brain and are thought to supply pathways guiding the migration of newly generated neurons (1, 5). Most of these mammalian radial glial cells disappear before birth (5) and process-bearing ependymal cells (tanycytes) have been reported to be present only in restricted portions of the adult brain (6).Using an antibod...