Abstract Rapidly advancing technology has resulted in the generation of the genomic sequences of several human tumors. These studies have revealed that tumors are heterogeneous and contain many mutations, supporting the mutator phenotype hypothesis of human cancer. Drivers of carcinogenesis are thought to be mutated genes that occur at high frequencies in large numbers of human tumors. However, functional characterization of many of the somatic mutations is lacking. We have identified several mutations of the DNA polymerase beta (POLB) gene in human colorectal cancer. Pol beta is the main polymerase involved in the base excision repair (BER) pathway and aberrant expression of Pol beta has been linked to a cancerous phenotype. In this study, we characterized the homozygous G231D mutation that was identified in a Stage 3 colorectal tumor. We have demonstrated that its expression in epithelial cells induced cellular transformation and increased the rate of proliferation. The transformed phenotype persisted in the cells even once the expression of G231D was extinguished. These data indicated that the expression of G231D caused an inheritable change; this change is most likely due to an increase in genomic instability caused by unfilled gaps proceeding through replication. Expression of G231D was insufficient to rescue Pol beta-deficient cells treated with chemotherapeutic agents suggesting that these agents may be effectively used to treat tumors harboring this mutation. Biochemical analysis of G231D revealed that it was 100-fold slower than WT Pol beta as determined by pre-steady state kinetics. The slow rate was a result of the decreased binding affinity of nucleotides by G231D. Residue 231 of Pol beta lies in close proximity to the template strand of the DNA. Molecular modeling showed that the change from a glycine to a negatively charged amino acid caused a repulsion between the template and residue 231 leading to the disruption of the dNTP binding pocket. Together, these data indicate that a person harboring the G231D Pol beta mutation may have an increased risk of cancer. Moreover, the altered sensitivity to chemotherapeutic drugs in cells carrying the G231D variant may have further implications in how to better improve the therapeutic outcome in patients with this mutation and supports the need for personalized cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B37.