Interferon (IFN) is being tested clinically in the treatment of a wide range of human malignancies. Patients undergoing cancer treatment may require radiotherapy in conjunction with IFN administration. This study examined the effect of purified preparations of IFN on the radiation response of mouse Swiss 3T3 cells in culture. Cells were exposed to 10 U/ml of mouse IFN or human IFN 2 hours prior to radiation. The IFN was left in the medium for the duration of the experiment. Marked enhancement of radiation response was observed in the presence of mouse IFN as compared to human IFN or no IFN treatment. The difference in radiation response was due to a reduction in the shoulder portion of the survival curve with no change in the slope of the exponential portion. Since human IFN was inactive in these experiments, the data suggest that the potentiation of radiation damage is specific for mouse IFN. The reduction in the shoulder in the presence of mouse IFN suggests the inhibition of the ability of cells to accumulate sublethal radiation injury. Split-dose experiments to test for sublethal radiation injury repair capacity failed to demonstrate an IFN effect. We conclude from these studies that IFN potentiates radiation injury. In clinical situations in which IFN is used, careful monitoring of dosage and timing of irradiation may be required to avoid excessive tissue damage. Moreover, treatment strategies may be directed to time radiation and IFN to obtain an improved therapeutic ratio.