We isolated spontaneously proliferating cells from primary astrocyte-enriched cultures prepared from neonatal rat brain. These cells proliferated and retained their characteristics for up to 50 generations. They expressed the microglial marker, OX42, but not glial fibrillary acidic protein, an astroglial marker. In addition, they possessed phagocytotic activity, and, when stimulated by lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma), they expressed proinflarnmatory mediators, including cytokines (i.e., interleukin (IL)- 1beta and tumor necrosis factor-alpha) and chemokines (i.e., IL-8 and monocyte chernotactic protein-1). Protein expression of inducible nitric oxide synthase and cyclooxygenase-2, and production of NO by these cells were induced by LPS or IFN-gamma. Thus, these cells possess the characteristics of microglia and can be used as a rat microglial cell line. (C) 2005 Elsevier Inc. All rights reserved.