DNA damage is a source of carcinogenicity and is also the source of the cytotoxicity of gamma-radiolysis and antitumor agents, such as the enediynes. The dioxobutane lesion (DOB) is produced by a variety of DNA-damaging agents, including the aforementioned. Repair of DOB is important for maintaining the integrity of the genome as well as counteracting therapeutic agents that target DNA. We demonstrate that the DOB lesion efficiently and irreversibly inhibits repair by DNA polymerase beta (Pol beta), an integral enzyme in base-excision repair. Irreversible inhibition of Pol beta by DOB suggests that this lesion provides a chemical explanation for the cytotoxicity of drugs that produce it and explains previously unexplained observations in the literature concerning abasic lesions that are not repaired efficiently. Finally, these observations provide the impetus for the design of a new family of inhibitors of Pol beta.