Phosphorothioate oligodeoxynucleotides with CpG motifs (CpG-ODNs) activate various immune cell subsets and induce production of numerous cytokines. To evaluate whether CpG-ODNs can induce rejection of established tumors, Lewis rats were inoculated intracerebrally with syngeneic CNS-1 glioma cells and subsequently injected with CpG-ODNs into the tumor bed. Although all of the control rats (n = 14) died within 23 days, 88% of the animals (n = 8) treated with a single CpG-ODN injection 5 days after tumor inoculation showed long-term survival (>90 days; P < 0.002). CpG-ODNs increased tumoral infiltration with macrophage/microglial cells, CD8, and natural killer lymphocytes. CpG-ODN-cured animals were further protected against a second tumor challenge. CpG-ODNs had no effect on a s.c. CNS1 tumor in nude mice, which suggested that CpG-ODN is not directly cytotoxic and that immunostimulation is required for the antitumoral effect. These findings suggest that intratumoral injections of CpG-ODNs represent a new immunotherapeutic approach in human gliomas, which overcome the need for the selection and purification of a tumoral antigen.