The tumor suppressor protein p53 has a critical role in safeguarding the integrity of the genome. Its functions are well understood but factors responsible for the transcriptional regulation of the p53 gene are almost entirely unknown. The DNA replication-related element (DRE)/DNA replication-related element-binding factor (DREF) transcriptional regulatory system is established as a master key to cell proliferation in Drosophila. DREF binds specifically to DRE sequences in the Drosophila p53 (dmp53) gene promoter as shown using anti-DREF antibodies in chromatin immunoprecipitation assays. Furthermore, a rough eye phenotype because of overexpression of DREF in Drosophila eye imaginal disks could be suppressed by half dose reduction of the dmp53 gene. In addition, the level of mRNA of dmp53 was decreased in DREF-knockdown cells and transient expression of the luciferase gene under control of the wild-type dmp53 gene promoter showed strong promoter activity in S2 cells, but this was almost completely abrogated with a DRE-mutated promoter. Requirement of DREs for dmp53 promoter activity was further confirmed by anti-beta-galactosidase antibody-staining of various tissues from transgenic flies carrying dmp53 promoter-lacZ fusion genes. These results indicate that DREF is necessary for dmp53 gene promoter activity.