To test the hypothesis whether DNA polymerases acquire mutator properties during tumor development (mutator hypothesis), we examined DNA polymerase δ mRNA in 6 colon cancer cell lines (DLD‐1, HCT116, SW48, HT29, SW480 and SW620) and 7 sporadic human colorectal cancers. For analysis we used amplification of cDNA by polymerase chain reaction, single‐strand conformation polymorphism and sequencing techniques. In 5 of the cell lines, 9 mutations leading to changes of the amino acid sequence of DNA polymerase δ were detected. Most mutations were found in the cell lines DLD‐1, HCT116 and SW48 for which defects in mismatch repair genes had been identified previously. In the majority of cases, wild type and mutated sequences were present. In 2 cell lines (HCT116 and SW48), a single‐nucleotide deletion occurred at the same position. This resulted in a premature termination codon by which the DNA interaction domain of the enzyme was eliminated. Furthermore, sequence deviations were found in the tumor tissues of 4 colon cancer patients. Wild‐type and altered sequences were present simultaneously. The deviations included missense mutations (2 cases) and silent mutations (2 cases). The missense mutations and one of the silent mutations were found in normal mucosa as well. In addition, the mutation clustered region of a tumor suppressor gene, often found to be defective in colon cancer, the adenomatous polyposis coli (APC) gene, was investigated in surgical specimens and cell lines. One carcinoma and 2 cell lines exhibited amino acid changes in both the DNA polymerase δ gene and in the mutation clustered region of the APC gene. Since most of the mutations detected in the DNA polymerase δ mRNA are likely to alter the structure of the protein, the enzyme is expected to be functionally impaired. In particular, copying fidelity might be decreased, thus contributing to the high mutation rate observed in colorectal cancer. Int. J. Cancer, 80:919–929, 1999. © 1999 Wiley‐Liss, Inc.