Genetic immunization against tumor antigens is an effective way to induce an immune response able to oppose cancer progression. Overexpression of HER-2/neu can lead to neoplastic transformation and has been found in many human primary breast cancers. We constructed DNA expression vectors encoding the full-length neu oncogene of rat cDNA (pCMV-NeuNT), the neu extracellular domain (pCMV-ECD), or the neu extracellular and transmembrane domains (pCMV-ECD-TM). We evaluated whether i.m. injection of these plasmids induces protection against the development of mammary tumors occurring spontaneously in FVB/N neu-transgenic mice. We found that pCMV-ECD-TM induced the best protection, whereas both pCMV-ECD and pCMV-NeuNT were less effective. The coinjection with a bicistronic vector for murine IL-12 increased the efficacy of pCMV-ECD and pCMV-NeuNT plasmids, and led to the same protection obtained with pCMV-ECD-TM alone. Anti-neuECD antibodies were detected in pCMV-ECD-TM vaccinated mice and, after coinjection with pCMV-IL12 plasmids, they appeared also in animals immunized with pCMV-ECD. Our data demonstrate the effectiveness of DNA vaccination using truncated Neu plasmids in inducing antitumor protection in a spontaneous mammary tumor model.