Macrophage-microglia were isolated from primary mixed brain cell cultures of normal newborn mice. They were successfully maintained in vitro for at least 8 weeks. Purity of the cultures was 97-100%, as determined by endocytosis of latex beads, non-specific staining through Fc receptors, EA and EAC rosette formation. These cells were non-specific esterase-positive, but peroxidase-negative. Electron-microscope observations revealed morphological similarities to mature macrophages. Isolated macrophage-microglia seldom incorporated [3H]thymidine in vitro. By means of 51Cr release assay, using monoclonal antibodies against mouse major histocompatibility complex (MHC) antigens and complement, we detected class I MHC (H-2) antigen on unstimulated macrophage-microglia, and both class I and class II (Ia) antigens on gamma-interferon-treated cells. These observations suggest possible immunoregulatory functions of macrophage-microglia in the central nervous system, as is characteristic of other cells of monocyte lineage.