The therapeutic efficacy of HER2/c-erbB-2/neu DNA immunization on mouse tumor cells expressing exogenous human or rat p185 n e u but not on mouse tumor cells naturally expressing mouse p185 n e u has been demonstrated. We investigated the feasibility of using N-terminal rat neu DNA immunization on mouse tumor overexpressing endogenous p185 n e u and enhancing the therapeutic efficacy of this vaccine by fusion to various cytokine genes, including interleukin-2 (IL-2), interleukin-4 (IL-4), or granulocyte-macrophage colony-stimulating factor. In a therapeutic model, N'-neu-IL-2 DNA vaccine was significantly better than N'-neu DNA vaccine, and N'-neu DNA vaccine was significantly better than control DNA or N'-neu-IL-4 DNA vaccine. The therapeutic efficacy of DNA vaccines was correlated with tumor infiltration of CD8 + T cells. Depletion of CD8 + T cells completely abolished the therapeutic effects of N'-neu-IL-2 DNA vaccine and N'-neu DNA vaccine. Depletion of CD4 + T cells after tumor implantation had no influence on N'-neu-IL-2 DNA vaccine, but enhanced the therapeutic efficacy of N'-neu DNA vaccine. Our results demonstrate that rat N'-neu DNA vaccine has a therapeutic effect on established tumor through the CD8 + T-cell-dependent pathway. Depletion of CD4 + T cells or fusion to the IL-2 gene can thus further enhance the therapeutic effects of N'-neu DNA immunization on mouse tumor expressing endogenous p185 n e u .