Vaccination with irradiated tumor cells genetically modified to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF tumor vaccine) induces a potent systemic antitumor immunity. To develop a protocol for cancer therapy to further augment the host immune response, we examined the effects of the GM-CSF tumor vaccines simultaneously producing additional cytokines. We prepared cancer vaccines expressing double cytokines by sequential recombinant retrovirus-mediated genetic transductions. We then used a murine intracerebral tumor model in which the GM-CSF tumor vaccine was less effective in immunopotentiation and evaluated tumor vaccines producing various cytokines in conjunction with GM-CSF. The cytokine combination of GM-CSF and interleukin 4 induced more potent antitumor immunity than GM-CSF alone. An in vivo depletion test showed that CD4⁺, CD8⁺, and asaloGM1⁺ cells were required for the optimum function of the GM-CSF plus interleukin 4 tumor vaccine. Histological examinations revealed infiltration of inflammatory cells at the site of tumor cell challenge as well as at the site of vaccination, indicating the induction of a systemic antitumor immune response which reached the central nervous system. Our findings suggest the feasibility of applying the intensified vaccination strategy to treat human cancers including malignant brain tumors.