p53 is a tumor suppressor gene found to be mutated in a wide variety of tumors. The encoded p53 protein has properties of a classical transcription factor, but the promoter targets for its regulation are largely unknown. We have investigated the ability of p53 to regulate activity of the human retinoblastoma susceptibility gene (Rb) promoter using a cotransfection assay in CCL-64 and Saos-2 cells. p53 was able to stimulate transcription from the Rb promoter at low input doses of p53 expression plasmid, whereas transcription was repressed at high input doses. The stimulatory effect of p53 on Rb promoter activity mapped to a region between 4 and 92 base pairs upstream from the start site of translation, whereas the region controlling repression by p53 mapped to the basal transcriptional control region of the promoter between -207 and -185. Moreover, an oligonucleotide containing Rb promoter sequences between -63 and -88 was sufficient to confer stimulation by p53 when inserted upstream from a minimal heterologous promoter. Gel mobility shift analysis was used to demonstrate that p53 can bind to a sequence within the -63 to -88 oligonucleotide with homology to a p53 binding site. The presence of a functional p53 binding site in the human retinoblastoma tumor suppressor gene promoter suggests that p53 can regulate Rb promoter activity.