A method has been developed to isolate brain macrophages (M phi) from normal neonatal and adult rats brain cell suspensions, as well as from brain cell suspensions of rat with experimental allergic encephalomyelitis (EAE), by making use of the ability of M phi to adhere to plastic surfaces. The isolated adherent cells were immuno- and enzyme-cytochemically identified. Phagocytic activity and the presence of Fc-IgG receptors were also examined. Approximately 30%-40% of the isolated adherent cells from neonatal rat brain are phagocytic and can be stained with macrophage-specific monoclonal antibodies, suggesting that these cells belong to the monocyte/macrophage lineage. From normal adult rat brain, only a small number of brain M phi could be isolated. A highly purified population of brain M phi was obtained from EAE rat brain. The isolated brain M phi are phagocytic, possess Fc-IgG receptors and rat M phi-associated antigens. Besides these features, the isolated brain M phi also express MHC class II antigens (Ia-antigens), which suggests that M phi may be involved in the regulation of immunological disorders of the CNS. The method reported here for rapidly isolating a large number of blood-monocyte-derived brain M phi from neonatal and adult brain allows an investigation of the precise role of M phi in inflammatory diseases of the central nervous system.