We examined cultures of purified type 1 astrocytes and mixed glial co-cultures containing type 1 astrocytes and 0-2A lineage cells in media containing fetal calf serum at 5 days in vitro (DIV), 12 DIV, and 30 DIV, using cell-specific immunocytochemical markers and electron microscopy. At all three time points and in both culture systems, the polygonal-shaped type 1 astrocytes were A2B5-, GFAP+, and GalC-(specific markers for 0-2A lineage cells, and mature astrocytes and oligodendrocytes, respectively). From 5 to 30 DIV, the type 1 astrocytes increased markedly in size and the appearance of the cytoskeleton changed dramatically, with the amount of glial filaments increasing and microtubules decreasing. At 5, 12, and 30 DIV, the 0-2A lineage cells were multipolar, A2B5 +, HNK-1 +, GFAP-, and GalC-. The 0-2 lineage cells could not be distinguished as either astrocytes or oligodendrocytes on the basis of immunocytochemical or ultrastructural characteristics. These cells had dense cytoplasm, very few intermediate filaments, and a large number of vacuoles and dense bodies. The general characteristics of the cultured astrocytes at 12 DIV and 30 DIV were similar to mature and aged astrocytes in vivo, respectively. These findings suggest that the culture environment in this study accelerated aging of type 1 astrocytes. 0-2A lineage cells, on the other hand, appeared unable to differentiate into either type 2 astrocytes or oligodendrocytes when cultured in the presence of both type 1 astrocytes and fetal calf serum.