We have documented previously that adenovirus-mediated interleukin 12 (IL-12) gene therapy is effective for orthotopic tumor control and suppression of pre-established metastases in a preclinical prostate cancer model (Y. Nasu et al., Gene Ther., 6: 338-349, 1999). In this report, we directly compare the effectiveness of an adenovirus that expresses both IL-12 and the costimulatory molecule B7-1 (AdmIL12/B7) with one that expresses IL-12 alone (AdmIL-12) using the poorly immunogenic RM-9 orthotopic murine model of prostate cancer. We document AdmIL-12/B7-mediated secretion of IL-12 and increased surface expression of B7-1 in infected RM-9 tumor cells. A significant reduction in orthotopic tumor size and increased survival was demonstrated in mice treated with a single orthotopic injection of AdmIL-12/B7 compared with AdmIL-12 or controls. Six of 19 animals treated with AdmIL-12/B7 survived long term with apparent eradication of the primary tumor in contrast to one of 38 animals in the AdmIL-12-treated group. Orthotopic treatment of tumors with both vectors led to an infiltration of both CD4+ and CD8+ immunoreactive cells, with AdmIL-12/B7 treatment having a more prolonged infiltration of CD8+ cells. AdmIL-12/B7 was also more effective than AdmIL-12 or controls at suppression of pre-established metastases. We further developed a vaccine model based on s.c. injection of infected, irradiated RM-9 cells and found that both AdmIL-12 and AdmIL-12/B7 are effective at suppressing the development and growth of challenge orthotopic tumors using this protocol.