The mutagenicity of cis-diamminedichloroplatinum(II) (DDP; cisplatin) and the rate at which resistance develops with repeated exposure to DDP are dependent on mutagenic translesional replication across DDP DNA adducts, mediated in part by DNA polymerase zeta, and on the integrity of the DNA mismatch repair (MMR) system. The aim of this study was to determine whether disabling Pol zeta by suppressing expression of its hREV3 subunit in human cancer cells can reduce the mutagenicity of DDP and whether loss of MMR facilitates mutagenic Pol zeta-dependent translesional bypass. The HCT116+ch3 (MMR(+)/REV3(+)) and HCT116 (MMR(-)/REV3(+)) human colon carcinoma cell lines were engineered to suppress hREV3 mRNA by stable expression of a short hairpin interfering RNA targeted to hREV3. The effect of knocking down REV3 expression was to completely offset the DDP resistance mediated by loss of MMR. Knockdown of REV3 also reduced the mutagenicity of DDP and eliminated the enhanced mutagenicity of DDP observed in the MMR(-)/REV3(+) cells. Similar results were obtained when the ability of the cells to express luciferase from a platinated plasmid was measured. We conclude that Pol zeta plays a central role in the mutagenic bypass of DDP adducts and that the DDP resistance, enhanced mutagenicity, and the increased capacity of MMR(-)/REV3(+) cells to express a gene burdened by DDP adducts are all dependent on the Pol zeta pathway.