To investigate the therapeutic efficacy of B7-1-expressing tumor vaccine on metastatic osteosarcoma, we introduced mouse B7-1 cDNA into a rat osteosarcoma cell line, MSK-8G. Flow cytometric analysis confirmed that the transfectants designated as B7-1-8G 10-1 and B7-1-8G 15-5 stably expressed B7-1 molecules on the cell surface. B7-1 transfectants were not only rejected by immunocompetent F344 rats but also conferred systemic immunity that protected against challenge with B7-negative parental osteosarcoma cells. In contrast, T-cell-deficient nude rats failed to reject B7-1 transfectants, indicating that T cells play a major role in the development of systemic immunity. We then conducted experimental therapies using irradiated B7-1-transfected tumor vaccine and methotrexate in an orthotopic implantation model. B7-1-transfected tumor vaccine significantly reduced the number of pulmonary metastatic nodules. Moreover, the combination of methotrexate and tumor vaccine further decreased the number of pulmonary metastatic nodules. Most important, the combined therapy with methotrexate and tumor vaccine resulted in a tumor-free condition as judged by the histopathological absence of tumors and survival of rats for more than 180 days. Furthermore, B7-1-transfected tumor vaccine could counteract the immunosuppressive effect of methotrexate. These findings strongly suggest that the B7-1-transfected tumor vaccine may be of clinical value for patients with metastatic osteosarcoma who exhibit immunosuppression due to chemotherapy.