Saccharomyces cerevisiae does not contain a p53 homolog. Utilizing this yeast as an in vivo test tube model, our aim was to investigate if a yeast protein would show p53 DNA binding homology. Electrophoretic mobility shift analyses revealed the formation of specific DNA-protein complexes consisting of S. cerevisiae nuclear protein(s) and oligonucleotides containing p53 DNA binding sites. A S. cerevisiae p53 binding site factor (Scp53BSF) bound to a p53 synthetic DNA-consensus sequence (SCS) and a p53 binding-site sequence from the MDM2 oncogene. The complexes were of comparable size. Like mammalian p53, the affinity of Scp53BSF for the SCS oligonucleotide was higher than for the MDM2 oligonucleotide. Binding of Scp53BSF to the SCS and MDM2 oligonucleotides was strongly competed by unlabeled oligonucleotides containing mammalian p53 sites, but very little by a mutated site oligonucleotide. Importantly, Scp53BSF-DNA binding activity was significantly induced in extracts from cells with DNA damage. This resulted in dose-dependent coordinated activation of transcription when using p53-binding site reporter constructs. An ancient p53-like DNA binding protein may have been found, and activation of DNA-associated factors to p53 response elements may have functions not yet determined.