Ammonium trichloro(dioxoethylene-o-o')tellurate (AS101) is a new synthetic compound previously described by us as being able to modulate the immune system and having minimal toxicity. Clinical trials are currently in progress with AS101 on cancer patients. AS101 has recently been found to have radioprotective effects on hemopoiesis and survival of irradiated mice when administered prior to irradiation. Radioprotection conferred by AS101 has recently been demonstrated by us to result partly from induction of progenitor cells to enter into S phase, which is assumed to be a more radioresistant phase of the cell cycle, and partly from the enhanced stimulation of CFU-S not only toward proliferation but also toward self-renewal. In the present study we demonstrate that the DNA repair processes expressing the cellular reponses associated with the restoration of the normal nucleotide sequence after damage caused to the DNA were also increased significantly after treatment with AS101. Unscheduled DNA repair synthesis was found to be significantly higher in both spleen and bone marrow cells from mice injected with AS101 compared to mice injected with PBS. DNA repair synthesis in spleen cells incubated with AS101 in vitro was also higher than that of PBS-treated cells. This was demonstrated by equilibrium alkaline cesium chloride density gradient of DNA from irradiated and nonirradiated spleen cells in the presence of hydroxyurea. In addition, using the neutral filter elution technique, we show that AS101 can both protect cells from DNA double-strand breaks (DSBs) induced by irradiation and enhance the ability of the affected cells to rejoin the DSBs. We show that extracts of splenocytes, either incubated with AS101 in vitro or obtained from mice injected with AS101, contain substantial DNA polymerase activity which is significantly higher compared to that of control treated cells. Aphidicolin, an inhibitor of DNA polymerases alpha and delta, and dideoxy-thymidine, an inhibitor of DNA polymerase beta, inhibited DNA repair synthesis of irradiated splenocytes stimulated with AS101. These results collectively indicate that AS101 confers its radioprotective effects partly by preventing the induction of DSBs induced by irradiation and partly by enhancing the ability of irradiated cells to repair their damaged DNA, probably by increasing mainly DNA polymerase activity. The understanding of the mechanism of radioprotection conferred by AS101 will enable us to use AS101 more effectively for the restoration of hemopoiesis in patients after radiation therapy or in patients suffering from overdose or accidental irradiation.