Expression of developmentally regulated antigens was used to characterize glial cells in cultures from embryonic mouse cerebral cortex. Over 90% of the cells had a flat morphology, and about 50% of these flat cells also expressed the ganglioside GD3. Up to 40% of all the GD3 expressing cells also expressed A2B5 antigen. Flat cells expressing either glial fibrillary acidic protein (GFAP), or GD3 or both were present at all times in vitro. These three populations of flat cells could not be further distinguished on the basis of NG2 or fibronectin expression, or with respect to their responses to the mitogens FGF-2, PDGF, or EGF. The glial cultures also contain a small number (approximately 5%) of process bearing cells with the morphological and immunocytochemical characteristics of oligodendrocyte precursors. The expression of GD3 by flat cells changed with time in culture as the fraction of flat cells expressing only GD3 declined and the fraction of cells expressing GFAP (with or without GD3) increased. The data are consistent with those flat cells expressing only GD3 being astrocyte precursors. Furthermore, between 1 and 3 weeks in vitro GD3/GFAP cells lose GD3 while retaining GFAP. Cells expressing only GFAP could be induced to express GD3 and A2B5 by treatment with FGF-2. The widespread and regulated expression of GD3 and A2B5 by murine glia is different from the restricted pattern of expression previously reported for these antigens in rat brain cell cultures. These results demonstrate that expression of GD3 and A2B5 by murine astrocytes depends on both culture age and extracellular signals and that these gangliosides are not markers for cell lineage in the mouse.