Synthetic oligodeoxynucleotides containing unmethylated CpG motifs detected by Toll-like receptor 9 of dendritic cells and B cells have potent immunomodulatory effects. CpG oligodeoxynucleotides induce cytokines, activate natural killer cells, and elicit T-cell responses leading to antitumor effects, including improved efficacy of chemotherapeutic agents and, as we reported recently, synergy between CpG oligodeoxynucleotide 1826 and single-dose radiotherapy of an immunogenic mouse fibrosarcoma. The present study extends this finding to the fractionated radiotherapy of the fibrosarcoma tumor and assesses the ability of CpG oligodeoxynucleotide 1826 to increase the radioresponse of a tumor (nonimmunogenic fibrosarcoma). The experiments used a murine immunogenic fibrosarcoma tumor, fibrosarcoma growing in the leg of mice, and response to radiotherapy was assessed by tumor growth delay and tumor cure rate (TCD₅₀, radiation dose yielding 50% tumor cure). Multiple s.c. peritumoral or i.t. administrations of CpG oligodeoxynucleotide 1826 at a dose of 100 μg per mouse were given when established tumors were 6 mm in diameter. Local tumor irradiation was initiated when tumors grew to 8 mm in diameter; radiation was delivered in 1 to 9 Gy fractions given twice daily separated by 6 to 7 hours for 5 consecutive days to achieve a total dose of 10 to 90 Gy. CpG oligodeoxynucleotide 1826, given as a single agent, had only a small antitumor effect, but it dramatically enhanced fibrosarcoma response to radiotherapy. Although 83.1 (79.2-90.0) Gy total dose were needed to achieve tumor cures in 50% of mice treated with radiotherapy alone, only 23.0 (11.5-32.7) Gy total dose were needed in mice treated with both CpG oligodeoxynucleotide 1826 and radiotherapy. The magnitude of potentiation of tumor radioresponse at the TCD₅₀ level was by a factor of 3.61, a much higher value than that (a factor of 1.93) that we reported for single-dose radiotherapy. Mice cured of their tumors by combined CpG oligodeoxynucleotide 1826 plus radiotherapy were highly resistant to s.c. tumor take or development of tumor nodules in the lung from i.v. injected tumor cells when rechallenged with fibrosarcoma cells 100 to 120 days after the treatment, suggesting the development of a memory response. CpG oligodeoxynucleotide 1826 also increased radioresponse of the nonimmunogenic fibrosarcoma tumor by a factor of 1.41 and 1.73 when CpG oligodeoxynucleotide 1826 was given s.c. and i.t., respectively. These findings show that CpG oligodeoxynucleotides are highly potent enhancers of tumor response to both single-dose and fractionated radiation and as such have potential to improve clinical radiotherapy.