As one of the three major replicative DNA polymerases, DNA polymerase ε (Pol ε) is uniquely positioned to exert a powerful effect on the kind and number of mutations introduced into the genome during each round of replication. Pol ε has two catalytic domains: a polymerase domain that selects strongly against mispaired deoxyribonucleotides, and an exonuclease domain that proofreads newly synthesized DNA and removes misinsertions. Tumors with mutations in the exonuclease domain of Pol ε also have unusually high genomic mutation frequencies, along with significantly higher numbers of C>A point mutations relative to tumors with wild-type Pol ε. Pol ε exonuclease active site mutants from Saccharomyces cerevisiae also demonstrate an increased tolerance for ribonucleotide incorporation during DNA synthesis. Ribonucleotides that persist in genomic DNA can lead to increased rates of mutagenesis. We sought to determine the extent to which human Pol ε exonuclease domain mutants contribute to genome instability through the incorporation of ribonucleotides. We show that human Pol ε mutants lacking 3'-5' exonuclease proofreading activity will incorporate ribonucleotides roughly 10 to 100 times more efficiently than yeast Pol ε, depending on the nature of the base. Exonuclease active site mutants are also more likely to allow misinserted deoxyribonucleotides to persist in newly synthesized DNA. We also sought to determine the relationship between the Pol ε cancer-associated exonuclease domain mutants and the observed C>A tumor mutation spectra. Pol ε cancer-associated mutants were more likely to make C>A mutations in vitro, although not at rates high enough to account for the C>A mutation rate in affected human tumors. These results suggest that Pol ε exonuclease domain mutations likely contribute to mutagenesis and tumorigenesis through multiple mechanisms in addition to base-base mispairs.