The transcription factor CCAAT/enhancer binding protein β (C/EBPβ) regulates the expression of key genes in inflammation but little is known about the involvement of C/EBPβ in glial activation. In this report, we have studied the patterns of astroglial and microglial C/EBPβ expression in primary mouse cortical cultures. We show that both astrocytes and microglia express C/EBPβ in untreated mixed glial cultures. C/EBPβ is upregulated when glial activation is induced by lipopolysaccharide (LPS). The LPS-induced upregulation of glial C/EBPβ is rapid (2 h at mRNA level, 4 h at protein level). It is elicited by low concentrations of LPS (almost maximal effect at 1 ng/mL) and it is reversed by the protein synthesis inhibitor cycloheximide. C/EBPβ nuclear levels increase both in astrocytes and microglia after LPS treatment, and the response is more marked in microglia. The LPS-induced increase in microglial C/EBPβ is prevented by coadministration of the MAP kinase inhibitors SB203580 (p38 inhibitor) + SP600125 (JNK inhibitor) or SB203580 + U0126 (ERK inhibitor). Systemic injection of LPS also increases brain nuclear levels of C/EBPβ as shown by Western blot, and this increase is localized in microglial cells as shown by double immunofluorescence, in the first report to our knowledge of C/EBPβ expression in activated glial cells in vivo. These findings support a role for C/EBPβ in the activation of astrocytes and, particularly, microglia. Given the nature of the C/EBPβ-regulated genes, we hypothesize that this factor participates in neurotoxic effects associated with glial activation. © 2006 Wiley-Liss, Inc.