Wild-type ErbB-2 (E2) positive D2F2/E2 tumors are rejected by active vaccination with ErbB-2 DNA. However, anti-ErbB-2 Ab response can cause cardiac toxicity or interfere with cellular immunity. It will be advantageous to induce only cellular immunity by active vaccination. A panel of E2 DNA vaccines were constructed, and their vaccination efficacy was ranked as E2> tyrosine kinase-deficient ErbB-2 (E2A)> full-length ErbB-2 targeted to the cytoplasm (cytE2)> tyrosine kinase-deficient cytE2 (cytE2A). E2A is a tyrosine kinase-deficient mutant containing a single residue substitution. CytE2 or cytE2A encodes a full-length protein that is targeted to and rapidly degraded in the cytosol by the proteasomes. Covaccination with cytE2A and GM-CSF or IL-2 DNA resulted in equivalent anti-tumor activity as E2. However, anti-ErbB-2 Ab was induced by E2 or E2A, but not cytE2 or cytE2A. Therefore, cytE2A appears to induce anti-tumor immunity without an Ab response. ErbB-2-specific CTL were detected in mice immunized with cytE2A and GM-CSF and have rejected tumor challenge. Depletion of CD8, but not CD4 T cells reduced anti-tumor immunity, indicating CTL as the effector cells. Covaccination with E2A and cytE2A induced synergistic anti-tumor activity, supporting enhanced peptide presentation from cytE2A, which was further evidenced by superior CTL activation using APCs expressing cytE2 vs E2. Taken together, cytoplasmic ErbB-2 DNA induced anti-tumor CTL, but not humoral response, demonstrating the feasibility of eliciting individual effector mechanism by targeted DNA vaccine.