Tamoxifen (TAM) possesses antiestrogen activity and is widely used for the treatment or prevention of breast cancer. However, it is also carcinogenic in human uterus and rat liver, highlighting the profound complexity of its actions. To explore the molecular mechanisms of TAM-induced mutagenesis, we analyzed the effects of this drug on gene-disrupted chicken B lymphocyte (DT40) clones deficient in various DNA repair pathways. Rad18, Rev3, and Polkappa are involved in translesion DNA synthesis (TLS), which facilitates recovery from replication blocks on damaged template strands. DT40 cells deficient in TLS were found to be hypersensitive to TAM, exhibiting an increase in chromosomal breaks. Furthermore, these mutants were also hypersensitive to 4-hydroxyestradiol, a physiological metabolite of estrogen. These data suggest a contribution of TLS to the prevention of chromosomal breaks by TAM and estrogen, and they therefore indicate that such error-prone DNA synthesis underlies mutagenesis induced by these agents.