The regulation of the C/EBP family in macrophages by LPS and cytokines is of potentially crucial importance in several pathophysiological conditions. The action of LPS and three cytokines on the expression of C/EBP mRNA, protein and functional DNA binding activity in the murine J774.2 cell line was therefore studied. Exposure of the cells to LPS, IL-1, IFN-γ and TNF-α produced a reduction of C/EBPα mRNA levels and a corresponding increase in the expression of C/EBPβ and C/EBPδ. EMSA showed time-dependent changes in the DNA binding activity of individual C/EBP isoforms and demonstrated the participation of heterodimers between the different members in DNA–protein interactions. Additionally, mediator-specific changes in the kinetics and magnitude of C/EBP mRNA expression pattern and profile of DNA-protein interactions were observed. These studies provide novel insights into the potential mechanisms that may be responsible for the mediator-specific regulation of macrophage gene expression through the C/EBP family.