Background: Amifostine is a potent scavenger of reactive oxygen species that is used for the salivary gland protection during therapy with radioactive iodine for thyroid cancer. There are no data on the potential effect of amifostine on thyroid cancer cells. Methods: We investigated the effects of the active form of amifostine (WR-1065) on the response of thyroid cancer cells to treatment with DNA-damaging agents. WR-1065 was examined in human thyroid cancer cell lines (FTC133, TPC1, BCPAP and C643) and embryonic fibroblast cells NIH3T3. DNA damage was induced by exposure to H2O2 (0.1 mM), by treatment with the radiomimetic neocarzinostatin (NCS 250 ng/mL) and by γ-radiation (6 Gy). DNA damage, cell viability and apoptosis were examined. Results: We demonstrated the selective action of WR-1065 (0.1 mM), which prevented oxidative stress–induced DNA damage in fibroblasts, but did not protect thyroid cancer cells from DNA damage and apoptosis documented by caspase-3 and PARP cleavage after exposure to H2O2, NCS and γ-radiation. Prolonged exposure to WR-1065 (0.1 mM for 24 h) was toxic for thyroid cancer cells; this treatment decreased the number of viable cells by 8% in C643 cells, 47% in TPC cells, 92% in BCPAP cells and 82% in FTC 133 cells. The cytotoxic effects of WR-1065 were not associated with induction of apoptosis. Conclusions: Our data show that amifostine has no protective effect on thyroid cancer cells against DNA-damaging agents in vitro and suggest that amifostine will not attenuate the efficacy of radioiodine treatment in patients with thyroid cancer.