Transcriptional regulation of the p53 gene plays an important role leading to elevated expression of mutant p53 alleles in tumor cells. In addition, alterations in p53 transcription levels occur in response to changes in the cell cycle. Previous work had identified a number of regulatory sites at the 5'-end of the murine p53 promoter. During the characterization of the 5'-end of the cloned murine p53 promoter, we identified a 28 bp positive regulatory element that participates in three distinct DNA-protein complexes. The binding by nuclear factors to each one of these sites contributes to the overall activity of the p53 promoter. One site is a potential recognition sequence for members of the ETS family of transcription factors, which are known regulators of the human p53 promoter. Since six nucleotides in the middle of this required element were not present in the previously published sequence of the murine promoter, we recloned this region from C57/BL6 cells and confirmed their presence in the genome. The removal of this regulatory element completely abolishes p53 promoter activity.