Purpose: The authors investigated whether a potential radioprotective effect of amifostine (WR-2721) after in vitro or in vivo administration can be detected with the comet assay. Moreover, it was determined whether radioprotection by WR-2721 is dependent on the concentration of amifostine or alkaline phosphatase (AP, the enzyme which activates the prodrug). Furthermore, the authors tried to detect possible interindividual differences in radioprotection by amifostine. Material and Methods: In vitro administration of amifostine: Freshly isolated lymphocytes from two healthy volunteers were incubated with different concentrations of AP (0-210 U/ml) and amifostine (0-5,000 μg/ml). In vivo administration of amifostine: Blood samples were collected from six postoperative rectal cancer patients before and after intravenous administration of amifostine 500 mg (no pretreatment with radio- or chemotherapy). Leukocytes and lymphocytes were irradiated and repaired in vitro and investigated with the alkaline comet assay. The radioprotective effect was evaluated by calculating dose-modifying factors (DMFs) and the paired t-test. Results: Amifostine alone did not alter the radiation-induced DNA damage in vitro. The addition of at least 0.5-1 U/ml AP was required. A significant radioprotective effect (p < 0.05) was seen after administration of amifostine in vitro for all concentrations investigated (250-5,000 μg/ml, initial DNA damage). A comparable radioprotective effect after in vivo administration of 500 mg amifostine was measured with a mean DMF of 0.87. Interindividual differences were present in vivo and in vitro. Conclusion: Amifostine 500 mg intravenously yields an adequate radioprotective concentration. The effect was only marginally improved by extreme concentrations of amifostine in in vitro experiments. The comet assay is capable of detecting small changes in radiosensitivity by amifostine.