The transcription factor CCAAT/enhancer binding protein-α (C/EBPα) can regulate the expression of important genes in the inflammatory response, but little is known about its role in glial activation. By using primary cortical murine glial cultures, we show that C/EBPα is expressed by microglial cells in vitro. Lipopolysaccharide (LPS) down-regulates C/EBPα mRNA at 2 hr and all C/EBPα protein isoforms at 4 hr. This effect is elicited by LPS concentrations ≥100 pg/ml. LPS-induced C/EBPα down-regulation occurs in microglial cells both in mixed glial and in microglial-enriched cultures. As seen with LPS, other toll-like receptor agonists (polyinosinic-polycytidylic acid, peptidoglycan from Staphylococcus aureus, and the oligonucleotide CpG1668) also down-regulate C/EBPα whereas cytokines such as interleukin-1β, interleukin-6, macrophage-colony stimulating factor, and interferon-γ do not. These findings suggest that C/EBPα down-regulation in activated microglia could play an important role in the increased expression of genes that are potentially pathogenic in a variety of neurological disorders. © 2007 Wiley-Liss, Inc.