A recently discovered potential tumor suppressor protein, Zac1, was previously shown to promote cell cycle arrest and apoptosis, and to act as a positive or negative transcriptional cofactor for nuclear receptors. Since these activities are common to Zac1 and p53, we tested for a functional interaction between these two proteins by investigating possible effects of Zac1 on the transcriptional activator function of p53. Zac1 specifically enhanced the activity of p53-responsive promoters in cells expressing wild type p53. The same promoters were not activated by Zac1 in cells lacking functional p53, but the Zac1 effect was restored by co-expression of p53. Zac1 bound to p53 and enhanced the activity of p53 or its N-terminal transcriptional activation domain fused to the DNA binding domain of Gal4. These results indicate that Zac1 served as a transcriptional coactivator for p53. The enhancement of p53 activity by Zac1 was much more dramatic in HeLa cells than in other cell lines tested. HeLa cells express human papillomavirus type 18 E6 protein which inactivates and causes the degradation of p53. Physical and functional interactions observed between Zac1 and E6 protein indicated that the dramatic activity of Zac1 in HeLa cells was due not only to Zac1's coactivator effect on p53, but also to the ability of Zac1 to reverse E6 inhibition of p53.