C/EBPβ is a member of the CCAAT/enhancer binding protein family of transcription factors and has been shown to be a critical transcriptional regulator of various proinflammatory genes, including IL-6 and MCP-1. Serine 64 in the transactivation domain of C/EBPβ has recently been identified as a Ras-induced phosphoacceptor site. The integrity of serine 64 along with threonine 189 is important for the Ha-ras^V12-induced transformation of NIH3T3 cells, however no target genes dependent upon serine 64 for their expression have been reported. In order to evaluate a potential role of serine 64 in C/EBPβ-regulated cytokine expression, we expressed a form of C/EBPβ with an alanine substitution at serine 64 (C/EBPβ_S64A) in P388 murine B lymphoblasts, which lack endogenous C/EBPβ expression and are normally unresponsive to LPS for expression of IL-6 and MCP-1. In comparison to wild type C/EBPβ, which robustly supports the LPS-induced expression of IL-6 and MCP-1, C/EBPβ_S64A was severely impaired in its ability to support the LPS-induced transcription of IL-6 and MCP-1. Furthermore, LPS stimulation increased the level of phosphorylation detected at serine 64. Thus, serine 64, probably through its phosphorylation, is a critical determinant of C/EBPβ activity in the transcription of IL-6 and MCP-1.