Macrophage inflammatory protein-2 (MIP-2) is a mouse C–X–C chemokine that plays an important role in the recruitment of neutrophils. Transcription of the MIP-2 gene is rapidly induced by lipopolysaccharide (LPS) stimulation in cells of macrophage lineage. We show here that the MIP-2 promoter is transcriptionally activated in a macrophage cell line RAW 264.7 by LPS through a sequence located between −450 and −54 and this region contains two copies of activator protein-1 (AP-1) and one copy of nuclear factor-κB (NF-κB) binding site. A MIP-2 promoter-reporter was activated by ectopical expression of NF-κB p65 or c-Jun transcription factors. Inhibition of NF-κB nuclear localization by co-expression of a mutant IκBα protein (IκBα super repressor, IκBαSR) blocked LPS-induced transcription from a MIP-2 promoter-reporter construct, showing that NF-κB activation is required for MIP-2 gene expression in the LPS-signaling pathway. By deletion analysis of the MIP-2 promoter region, we show that NF-κB and c-Jun binding sites are essential for LPS-induced MIP-2 gene expression. Using transient transfection, NF-κB and c-Jun transcription factors were found to synergistically activate the MIP-2 promoter. In summary, our data suggest that both NF-κB and c-Jun contribute to LPS-induced mouse MIP-2 gene expression in RAW 264.7 cells.