Cdc25A is a tyrosine phosphatase that is involved in the regulation of the G1/S phase transition by activating cyclin E/Cdk2 and cyclin A/Cdk2 complexes through removal of inhibitory phosphorylations. The E6 and E7 oncoproteins of the high-risk human papillomaviruses (HPV) interact with and functionally abrogate the p53 and pRB proteins, respectively. In the present study we have investigated the regulation of the Cdc25A promoter during G1 and S-phases of the cell cycle and by the HPV-16 E7 oncoprotein. Serum induction leads to a derepression of the Cdc25A promoter and can be mediated through two E2F binding sites, E2F-A and E2F-C. While E2F-A is by both E2F1 and E2F4, E2F-C is regulated only by E2F1. The Cdc25A promoter is transactivated by the E7 oncogene of HPV-16. Furthermore, Cdc25A levels are highly increased in E7-expressing cell lines. Inducible expression of E7 leads to an immediate increase in Cdc25A protein levels. These data suggest that Cdc25A may be a critical target of HPV-16 E7 in the disruption of the G1/S phase transition.