The base excision repair pathway (BER) is believed to maintain genomic integrity by repairing DNA damage arising spontaneously or induced by oxidizing and alkylating agents. To establish the role of DNA polymerase β (β-pol) in BER and β-pol-dependent BER in maintaining genomic stability, we have measured the impact of a gene-targeted disruption in the β-pol gene on DNA repair capacity and on in vivo sensitivity to carcinogens. We have extensively phenotyped the DNA β-pol heterozygous (β-pol^+/−) mouse as expressing ∼50% less β-pol mRNA and protein and as exhibiting an equivalent reduction in the specific activity of β-pol. We measured BER activity by in vitro G:U mismatch and ^8-OHG:C repair and find that there is a significant reduction in the ability of extracts from β-pol^+/− mice to repair these types of DNA damage. In vivo, the β-pol^+/− mice sustain higher levels of DNA single-strand breaks as well as increased chromosomal aberrations as compared with β-pol^+/+ littermates. Additionally, we show that reduction in β-pol expression and BER activity results in increased mutagenicity of dimethyl sulfate as evidenced by a 2-fold increase in LacI mutation frequency. Importantly, the β-pol^+/− mice do not exhibit increased sensitivity to DNA damage induced by N-nitroso-N-ethylurea, ionizing radiation, or UV radiation, which induce damage processed by alternative repair pathways, demonstrating that this model is specifically a BER-deficient model.