An active immunotherapeutic strategy using transfected allogeneic cells for targeting the mutant epidermal growth factor receptor (EGFRvIII) on intracranial tumors was examined. Immunization with allogeneic 300.19/EGFRvIII cells induced CD8+ cytotoxic T-lymphocytes against EGFRvIII bearing syngeneic B16-F10 melanoma or 560 astrocytoma cells (H-2b), but not against allogeneic NR6 cells (H-2q) also bearing EGFRvIII significant NK cell activity was also noted in vitro. Vaccination protected against intracranial challenge with EGFRvIII-positive tumor, with 50% long term survival. In vivo depletions of effector cell subsets demonstrated the requirements for both CD8+ and CD4+ T-cells but not NK cells in producing this protective effect. These data demonstrate the generation of significant, antigen-specific and MHC class I-restricted cytotoxic immune responses which are effective against tumors present in the CNS.