Microglia can be isolated with high purity but low yield by shaking off loosely adherent cells from mixed glial cultures. Here we describe a new technique for isolating microglia with an average yield close to 2,000,000 microglial cells/mouse pup, more than five times higher than that of the shaking method. Confluent mixed glial cultures are subjected to mild trypsinization (0.05–0.12%) in the presence of 0.2–0.5 mM EDTA and 0.5–0.8 mM Ca2+. This results in the detachment of an intact layer of cells containing virtually all the astrocytes, leaving undisturbed a population of firmly attached cells identified as > 98% microglia. These almost pure microglial preparations can be kept in culture for weeks and show proliferation and phagocytosis. Treatment with macrophage colony-stimulating factor and lipopolysaccharide, alone or in the presence of interferon γ, induces typical microglial responses in terms of proliferation, morphological changes, nuclear factor-κB translocation, NO, and tumor necrosis α release and phagocytosis. This method allows for the preparation of highly enriched mouse or rat microglial cultures with ease and reproducibility. Because of its high yield, it can be especially convenient when high amounts of microglial protein/mRNA are required or in cases in which the starting material is limited, such as microglial cultures from transgenic animals. © 2003 Wiley-Liss, Inc.