The CXC chemokine, melanoma growth stimulatory activity/growth-regulated protein, CXCL1 is an important modulator of inflammation, wound healing, angiogenesis, and tumorigenesis. Transcription of CXCL1 is regulated through several cis-acting elements including Sp1, NF-kappa B, and an element that lies immediately upstream of the NF-kappa B element, the immediate upstream region (IUR). A transcription element data base search indicated that the IUR element contains a binding site for the transcriptional repressor, human CUT homeodomain protein/CCAAT displacement protein (CDP). It is shown here that in electrophoretic mobility shift assays, complexes obtained with the IUR oligonucleotide probe are supershifted by anti-CDP antibodies and that a CDP polypeptide containing a high affinity DNA binding domain binds to the sequence GGGATCGATC in the IUR element. In Southwestern blot analyses, oligonucleotides containing the wild-type IUR sequence, but not a mutant oligonucleotide with substitutions in the GGGATCGATC sequence, bind a 170--180-kDa protein. Furthermore, overexpression of the CDP protein blocks CXCL1 promoter activity in reporter gene assays, whereas overexpression of an antisense CDP construct leads to a significant increase in CXCL1 promoter activity. Mutations in the IUR element, which map in the putative CDP-binding site, inhibit the binding of CDP to the IUR element and favor increased transcription from the CXCL1 promoter. Based on these results, we propose that transcriptional regulation of the CXCL1 gene is mediated in part by CDP, which could play an important role in inflammatory processes and tumorigenesis.