FVB/N mice transgenic for nontransforming rat neu develop spontaneous breast cancers that are neu positive and estrogen receptor negative, mimicking premenopausal human breast cancer. These animals have been widely used as a model for immunobased therapies targeting HER-2/neu. In this study, we used serological analysis of recombinant cDNA expression libraries to characterize the antigenic repertoire of neu transgenic (neu-tg) mice and questioned the ability of this murine model to predict potential human tumor antigens. After screening 3 x 10(6) clones from 3 different cDNA libraries, 15 tumor antigens were identified, including cytokeratin 2-8, glutamyl-prolyl-tRNA synthetase, complement C3, galectin 8, and serine/threonine-rich protein kinase 1. Multiple proteins involved in the Rho/Rho-associated, coiled coil-containing protein kinase (Rock) signal transduction pathway were found to be immunogenic, including Rock1, Rho/Rac guanine nucleotide exchange factor 2, and schistosoma mansoni adult worm antigen preparation 70. All of the identified antigens are self-proteins that are expressed in normal tissues in addition to breast tumors and the majority of the antigens are intracellular proteins. More than half of the mouse tumor antigens have human homologues that have been reported previously as tumor antigens. Finally, the tumor-specific antibody immunity and marked immune cell infiltration that was observed in mice with spontaneous tumors were not observed in mice with transplanted tumors. Our results indicate that neu-tg mice bearing spontaneous tumors develop humoral immunity to their tumors similar to cancer patients and that tumor antigens identified in transgenic mouse may predict immunogenic human homologues.