To protect genome integrity and ensure survival, eukaryotic cells exposed to genotoxic stress cease proliferating to provide time for DNA repair. Human cells responded to ultraviolet light or ionizing radiation by rapid, ubiquitin- and proteasome-dependent protein degradation of Cdc25A, a phosphatase that is required for progression from G1 to S phase of the cell cycle. This response involved activated Chk1 protein kinase but not the p53 pathway, and the persisting inhibitory tyrosine phosphorylation of Cdk2 blocked entry into S phase and DNA replication. Overexpression of Cdc25A bypassed this mechanism, leading to enhanced DNA damage and decreased cell survival. These results identify specific degradation of Cdc25A as part of the DNA damage checkpoint mechanism and suggest how Cdc25A overexpression in human cancers might contribute to tumorigenesis.