AbstractThe aim of the present study was to prepare cultures enriched in type‐2 astrocytes (AS) and to analyze some of the properties of these cells over relatively long culture periods. Cultures enriched in type‐2 AS were obtained by subculturing, at low cell density and in the presence of fetal calf serum, a cell population containing numerous bipotential glial precursors. This cell population was detached mechanically from 2‐ to 3‐week primary mixed glial cultures prepared from 1‐day postnatal rat cerebral cortex. The cellular composition of the subcultures was analyzed immunocytochemically over a period of 3 weeks using various combinations of antibodies, recognizing a set of differentiated and a set of undifferentiated glial antigens (glial fibrillary acidic protein [GFAP], galactocerebroside, sulfatide, gangliosides binding the monoclonal antibodies A2B5 and LB1, fibronectin). Most LB1+, A2B5+glial precursors differentiated into type‐2 AS within a week. At this stage, type‐2 AS accounted for more than 70% of cells in the cultures and exhibited the characteristic features previously described for these cells (stellate shape, GFAP, LB1 and A2B5 positivity, ability to accumulate [3H]GABA and to synthesize chondroitin sulfate, low proliferative activity). About one third of the type‐2 AS also were recognized by O4 (antisulfatide) antibodies. The major contaminants were macrophages (10–15%) and fibroblastic cells (5–10%). In longer term cultures, type‐2 AS tended to lose several of these features. Many acquired a flat, polygonal shape and lost LB1 positivity. The ability to accumulate [3H]GABA progressively decreased, as did the expression of chondroitin sulfate, although to a lesser degree. Although losing several of their properties, type‐2 AS did not appear to acquire the properties of type‐1 AS: their proliferative activity remained very low, and they did not express class II antigens of the major histocompatibility complex upon stimulation with γ‐interferon. Some bec