The chicken DT40 B lymphocyte line diversifies its immunoglobulin (Ig) V genes through translesion DNA synthesis-dependent point mutations (Ig hypermutation) and homologous recombination (HR)-dependent Ig gene conversion. The error-prone biochemical characteristic of the A family DNA polymerases Polnu and Pol led us to explore the role of these polymerases in Ig gene diversification in DT40 cells. Disruption of both polymerases causes a significant decrease in Ig gene conversion events, although POLN(-/-)/POLQ(-/-) cells exhibit no prominent defect in HR-mediated DNA repair, as indicated by no increase in sensitivity to camptothecin. Poleta has also been previously implicated in Ig gene conversion. We show that a POLH(-/-)/POLN(-/-)/POLQ(-/-) triple mutant displays no Ig gene conversion and reduced Ig hypermutation. Together, these data define a role for Polnu and Pol in recombination and suggest that the DNA synthesis associated with Ig gene conversion is accounted for by three specialized DNA polymerases.