Bypass of the configurationally stable analogue (β-C-Fapy·dG) of the formamidopyrimidine lesion derived from 2′-deoxyguanosine oxidation (Fapy·dG) was studied in vitro and in Escherichia coli. The exonuclease deficient Klenow fragment of E. coli DNA polymerase I (Klenow exo −) misincorporated dA most frequently opposite β-C-Fapy·dG, but its efficiency was <0.2% of dC insertion. Klenow exo − fidelity was enhanced by the enzyme’s high selectivity for extending duplexes only when dC was opposite β-C-Fapy·dG. The expectations raised by these in vitro data were realized when β-C-Fapy·dG replication was studied in E. coli by transfecting M13mp7(L2) bacteriophage DNA containing the nucleotide analogue within the lacZ gene in 4 local sequence contexts. The bypass efficiency of β-C-Fapy·dG varied between 45% and 70% compared to a genome containing only native nucleotides. Mutation frequencies at the site of the lesions in the originally transfected genomes were determined using the REAP assay [Delaney, J. C.; Essigmann, J. M. Methods Enzymol. 2006, 408, 1]. The levels of mutations could not be distinguished between those observed when genomes containing native nucleotides were replicated, indicating that the mutagenicity of β-C-Fapy·dG was <1%. These data and previous reports indicate that β-C-Fapy·dG is a good model of Fapy·dG in E. coli. In addition, these results and the previous report of β-C-Fapy·dG binding to the base excision repair protein formamidopyrimidine glycosylase suggest that this analogue could be useful as a DNA repair inhibitor.