Several low fidelity DNA polymerases participate in generating mutations in immunoglobulin genes. Polymerase η is clearly involved in the process by causing substitutions of A:T base pairs, whereas polymerase ι has a controversial role. Although the frequency of mutations was decreased in the BL2 cell line deficient for polymerase ι, hypermutation was normal in the 129 strain of mice, which has a natural nonsense mutation in the Poli gene. It is possible that the mice compensated for the defect over time, or that polymerase η substituted in the absence of polymerase ι. To examine polymerase ι in a genetically defined background, we backcrossed the 129 nonsense mutation to the C57BL/6 strain for six generations. Class switch recombination and hypermutation were studied in these mice and in congenic mice doubly deficient for both polymerases ι and η. The absence of both polymerases did not affect production of IgG1, indicating that these enzymes are not involved in switch recombination. Poli-/-F6 mice had the same types of nucleotide substitutions in variable genes as their C57BL/6 counterparts, and mice doubly deficient for polymerases ι and η had the same mutational spectrum as Polh-/- mice. Thus, polymerase ι did not contribute to the mutational spectra, even in the absence of polymerase η.