Oligodeoxynucleotides containing CpG motifs (CpG-ODN) display broad immunostimulating activity and are currently under clinical trial in various malignancies, including recurrent glioblastomas. Combining CpG-ODN with another therapy that could induce antigen release might enhance tumor-specific immune response. We investigated whether radiotherapy (RT) could be associated advantageously to intratumoral injections of CpG-ODN. Fisher rats bearing 9L glioma were treated with various combinations of RT and CpG-28, an oligonucleotide with good immunostimulating activity. RT and CpG-28 induced complete tumor remission in one-third of the animals. When both treatments were combined, complete tumor remission was achieved in two-thirds of the animals (p < 0.001 when compared to non-treated rats, p < 0.03 when compared to CpG-28 alone). Such efficacy was not observed in nude mice, underlying the role of T cells in antitumor effects. The combination of both treatments appeared optimal when the delay between RT and CpG-28 administration was <3 days (from 100% survival for a 3 days delay, to 57% survival for a 21 days delay, p < 0.05). Tumor infiltration by immune cells and expression within tumors of the CpG receptor, TLR9, were not modified by irradiation. These results support an attractive strategy of sequential radiotherapy and immunotherapy by CpG-ODN and have potential implications for future clinical trials with CpG-ODN. © 2005 Wiley-Liss, Inc.