It is believed that ramified resting microglial cells in the brain are differentiated from macrophage-like ameboid cells, although the mechanism for the differentiation is not fully understood. In the present study, we investigated whether the differentiation of microglial cells is observable in mixed brain cell culture prepared from newborn rat forebrains. In confluent mixed brain cell culture, both ramified and ameboid microglial cells were simultaneously present. The ramified cells were located in or under the astrocyte monolayer, while the ameboid cells were over the layer as revealed by confocal laser scan microscopy. The majority of ramified cells appeared after the astrocyte layer was completely formed and they downregulated the expression of the major histocompatibility complex antigen. Fibronectin was detected around ramified microglial cells, and laminin was also present in the astrocyte monolayer in mixed brain cell culture, while both proteins were not distributed near ameboid cells over the monolayer. When purified microglial cells were cultured on astrocyte-derived extracellular matrix in serum-free medium, they ramified. These results show that the differentiation of microglial cells is observable in culture and that astrocytes may play pivotal roles in the differentiation mainly by secreting insoluble factors.