The mechanisms responsible for creating genetic errors and genomic instability in cancer cells have not been fully defined. Recently, it has been shown that human cells contain a highly organized complex of proteins, termed the DNA synthesome, that is fully competent to carry out all phases of SV40 in vitro DNA replication (J. M. Coll et al, Oncol. Res., 8: 435-447, 1996; L. H. Malkas et al., Biochemistry, 29: 6362-6374, 1990; Y. Wu et al., J. Cell. Biochem., 54: 32-46, 1994; N. Applegren et al., J. Cell. Biochem., 54: 32-46, 1994). DNA replication fidelity analyses of the DNA synthesome derived from malignant and nonmalignant human breast cells demonstrate that the malignant cell synthesome is mutagenic. The decrease in tumor cell replication fidelity was not due to an increased proliferative capacity of the tumor cells or an increase in the synthetic activity of their DNA synthesome. The ratios of insertions, deletions, and mismatches created by the synthesome from malignant and nonmalignant breast cells were essentially identical, despite the greater overall number of mutations made by the breast cancer cell synthesome. These data define, for the first time, a mechanism unique to cancer cells that contributes to the observed increase in genetic mutation in cancer cells.