Mammalian DNA polymerase (Pol) delta is essential for DNA replication. It consists of four subunits, p125, p50, p68, and p12. We report the discovery that the p12 subunit is rapidly degraded in cultured human cells by DNA damage or replication stress brought about by treatments with UV, methyl methanesulfonate, hydroxyurea, and aphidicolin. The degradation of p12 is due to an accelerated rate of proteolysis that is inhibited by the proteasome inhibitors, MG132 and lactacystin. UV treatment converts Pol delta in vivo to the three-subunit form lacking p12. This was demonstrated by its isolation using immunoaffinity chromatography. The three-subunit enzyme retains activity on poly(dA)/oligo(dT) templates but is impaired in its ability to extend singly primed M13 templates, clearly indicating that its in vivo functions are likely to be compromised. This transformation of Pol delta by modification of its quaternary structure is reversible in vitro by the addition of the p12 subunit and could represent a novel in vivo mechanism for the modulation of Pol delta function. UV and hydroxyurea-triggered p12 degradation is blocked in ATR(-/-) cells but not in ATM(-/-) cells, thereby demonstrating that p12 degradation is regulated by ATR, the apical kinase that regulates the damage response in S-phase. These findings reveal a novel addition to the cellular repertoire of DNA damage responses that also impacts our understanding of the role of Pol delta in both DNA replication and DNA repair.