The Cdc25 A phosphatase is required for the G 1–S transition of the cell cycle and is overexpressed in human cancers. We found that it is ubiquitylated and rapidly degraded by the proteasome and that its levels increase from G 1 until mitosis. By treating cells with the DNA synthesis inhibitor hydroxyurea, Cdc25 A rapidly decreased in abundance, and this was accompanied by an increase in Cdk2 phosphotyrosine content and a decrease in Cdk2 kinase activity. Cdc25 A overexpression altered the ability of cells to arrest in the presence of hydroxyurea, and caused them to undergo premature chromosome condensation. Cdc25 A overexpression could render tumor cells less sensitive to DNA replication checkpoints, thereby contributing to their genomic instability.