Ref-1 is a multifunctional protein that stimulates DNA binding by a number of transcription factors and serves as the abasic (A/P) endonuclease in base excision repair. Ref-1 was discovered to be a potent activator of p53 DNA binding in vitro. To address the physiological significance of the effects of Ref-1 on p53, we have analyzed its role in regulating p53 function in vivo. We found that Ref-1 over-expression enhances the ability of p53 to transactivate a number of p53 target promoters and increases the ability of p53 to stimulate endogenous p21 and cyclin G expression. Additionally, it was observed that Ref-1 associates with p53 in vivo and in vitro. Importantly, downregulation of Ref-1 (by antisense) causes a marked reduction in p53 induction of p21 mRNA and protein, as well as diminished ability of p53 to transactivate the p21 and Bax promoters. Moreover, Ref-1 levels are correlated with the extent of apoptosis induced by p53. Finally, we observed that Ref-1 cooperates with a DNA-damaging compound, camptothecin, to stimulate the transcriptional activity of p53. Together these data indicate that Ref-1 is a key cellular regulator of p53.