Accurate DNA synthesis by the replicative DNA polymerases α, δ, and ε is critical for genome stability in eukaryotes. In humans, over 20 SNPs were reported that result in amino–acid changes in Polδ or Polε. In addition, Polδ variants were found in colon–cancer cell lines and in sporadic colorectal carcinomas. Using the yeast-model system, we examined the functional consequences of two cancer-associated Polδ mutations and four polymorphisms affecting well-conserved regions of Polδ or Polε. We show that the R696W substitution in Polδ (analog of the R689W change in the human cancer-cell line DLD-1) is lethal in haploid and homozygous diploid yeast. The cell death results from a catastrophic increase in spontaneous mutagenesis attributed to low-fidelity DNA synthesis by Polδ-R696W. Heterozygotes survive, and the mutation rate depends on the relative expression level of wild-type versus mutant alleles. Based on these observations, we propose that the mutation rate in heterozygous human cells could be regulated by transient changes in gene expression leading to a temporary excess of Polδ-R689W. The similarities between the mutational spectra of the yeast strains producing Polδ-R696W and DLD-1 cells suggest that the altered Polδ could be responsible for a significant proportion of spontaneous mutations in this cancer cell line. These results suggest that the highly error-prone Polδ-R689W could contribute to cancer initiation and/or progression in humans.