ICAM-1 is an inducible cell surface protein that is involved in cell extravasation into inflamed tissues as well as immune responses. ICAM-1 expression is upregulated by proinflammatory cytokines such as TNF-alpha and IL-1beta in numerous cell types including the astrocyte, which functions as an immune effector cell in the central nervous system (CNS). We investigated the mechanism by which the ICAM-1 gene is transcriptionally regulated in astrocytes in response to TNF-alpha and IL-1beta. Human ICAM-1 promoter constructs linked to the reporter gene luciferase were transiently transfected into astrocytes, stimulated with TNF-alpha and IL-1beta, and ICAM-1 promoter activity examined. We determined that binding sites for both NF-kappaB (-186 bp region) and C/EBP (-198 bp region) are involved in TNF-alpha and IL-1beta-mediated ICAM-1 upregulation. Electrophoretic mobility shift assays using antibodies against NF-kappaB and C/EBP isoforms showed that p65 homodimers and p65/p50 heterodimers bind to the NF-kappaB site, and C/EBPdelta homodimers and C/EBPbeta/delta heterodimers bind to the C/EBP site. Transient transfection assays demonstrated that overexpression of p65 could transactivate the promoter activity of ICAM-1 reporter constructs. p50 overexpression had no effect on the basal levels of ICAM-1 transcription, but inhibited, in a dose dependent manner, p65 mediated transcription. Overexpression of C/EBPbeta slightly inhibited basal levels of ICAM-1 promoter activity, however, when C/EBPbeta and p65 were cotransfected, C/EBPbeta completely abolished the transactivating effects of p65. These results demonstrate that cytokine-induced ICAM-1 expression in astrocytes is regulated by interactions between NF-kappaB and C/EBP transcription factors.