We have previously shown that enriched preparations of oligodendrocytes from either mature bovine brain or 30-d-old rat brain, when cultured in serum-free medium, yield mixed cultures of oligodendrocytes and astrocytes even though no GFAP+ cells were present after 24 hr in culture (Norton et al., 1986, 1988). To test the possibility that the astrocytes in these cultures arose from glial precursor cells, we followed the expression of ganglioside GD3, galactosylceramide (GC), glial fibrillary acidic protein (GFAP), and vimentin in the cultures. GD3 has already been shown to be a marker of immature neuroectodermal cells, which in the postnatal brain are glial progenitor cells (Goldman et al., 1984, 1986). The cultures from both species contained at 1 DIV only two populations of cells; 90-95% GC+/GD3- oligodendrocytes and 4-10% GD3+/GC- small, round cells. With time, the oligodendrocytes remained GD3-/GFAP-/vimentin-. The kinetics of antigen expression of the GD3+ cells could best be interpreted by the following sequence: (sequence; see text) We interpret these results to show that the astrocytes arose from a small population of GD3+ glial precursor cells present in the brain that were co-isolated with oligodendroglia. No evidence was obtained that these GD3+ cells could also differentiate into oligodendrocytes.