The 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 gamma-interferon. Some became positive for fibronectin.