Multiple DNA polymerases are involved in the generation of somatic mutations during Ig gene hypermutation. Mice expressing a catalytically inactive REV1 (REV1AA) exhibit reduction of both C to G and G to C transversions and moderate decrease of A/T mutations, whereas DNA polymerase η (POLH) deficiency causes greatly reduced A/T mutations. To investigate whether REV1 and POLH interact genetically and functionally during Ig gene hypermutation, we established REV1AA Polh−/− mice and analyzed Ig gene hypermutation in the germinal center (GC) B cells. REV1AA Polh−/− mice were born at the expected ratio and developed normally with no apparent gross abnormalities. B-cell development, maturation, Ig gene class switch and the GC B-cell expansion were not affected in these mice. REV1AA Polh−/− B cells also exhibited relatively normal sensitivity to etoposide and ionizing radiation. Analysis of somatic mutations in the JH4 intronic region revealed that REV1AA Polh−/− mice had a further decrease of overall mutation frequency compared with REV1AA or Polh−/− mice, indicating that the double deficiency additively affected the generation of mutations. Remarkably, REV1AA Polh−/− mice had nearly absent C to G and G to C transversions, suggesting that POLH is essential for the generation of residual C to G and G to C transversions observed in REV1AA mice. These results reveal genetic interactions between REV1 catalytic activity and POLH and identify an alternative pathway, mediated by non-catalytic REV1 and POLH, in the generation of C to G and G to C transversions.