Ganglioside GD3 occurs in immature cells in the neuroectoderm. However, with regard to particular cellular locations of GD3, rat brain has received more attention than mouse brain. In brains from neonatal mice the most intense GD3 immunostaining appears to occur in structures that differ from those that immunostain the most intensely in brains from neonatal rats (Cammer and Zhang: J Histochem Cytochem 44: 143-149, 1996). In the present study epifluorescence and confocal microscopy were used for the purpose of identifying the types of GD3-immunopositive structures in brains of neonatal, 2-week-old, and adult mice. Vibratome sections from mouse brains were double immunostained for GD3 and respective markers for macrophages, microglia, and cells belonging to the oligodendrocyte lineage. Surprisingly, none of those marker antigens immunostained intensely in the same respective structures as GD3. The GD3-positive structures, however, did resemble protoplasmic astrocytes and radial glia, some with GD3-positive end-feet at the glia limitans; however, we did not rule out the possibility that there might be some GD3 on the surfaces of prooligodendroblasts. The scarcity of glial fibrillary acidic protein (GFAP)-positive cells in brains of neonatal mice made it impractical to look for GD3+/GFAP+ structures that might belong to the astrocyte lineage. The Mu subunit of glutathione-S-transferase (Mu) was shown to label radial glia and the few GFAP-positive cells in brains of neonatal mice. Subsequently, confocal microscopy showed Mu and GD3 to be colocalized in radial glia and protoplasmic astrocytes in the neonate. In brains from mice > or = 2 weeks of age GD3 immunostaining was demonstrated in GFAP-positive astrocytes, including reactive astrocytes. Much of the GD3 appeared to occur at the tips of astrocyte processes. It is suggested that GD3 in radial glia and astrocytes may function as a ligand enabling recognition of those structures by neurons or as a precursor of more complex gangliosides in neurons.