Recombinant viruses can produce cytokines in tumors mobilizing an immune response to tumor cells. In this study, the authors investigated gene expression, in vivo antitumor efficacy, and safety of attenuated recombinant vaccinia virus (rVV) carrying murine cytokine genes interleukin (IL)-2 (rVV–mIL-2), IL-12 (rVV–mIL-12), and both IL-2 and IL-12 (rVV-2–12) in an athymic nude mice model. Significant tumor inhibition (p < 0.05) was observed in a preestablished subcutaneously implanted C6 glioma model using rVVs at doses ranging from 102to 107plaque forming units (PFU). An antitumor effect did not depend on the dose of the rVV–mIL-2 and rVV–mIL-12 viruses. All constructed rVVs induced a high level of cytokine expression in vitro and in vivo. Most groups injected with high doses of recombinant viruses encoding cytokine genes (105to 107PFU) showed signs of cytokine toxicity, whereas in the low-dose treatment groups (102to 103PFU) toxicity was greatly reduced. The antitumor activity of rVV–mIL-12 was associated with increases in both the percentage and number of natural killer T cells in the spleen. Local detection of interferon-&ggr; and tumor necrosis factor-&agr; was also correlated with tumor growth arrest induced by the treatment. High-dose VV control vector per se induced tumor inhibition by activating Mac-1+cells in blood, but the antitumor effect was less pronounced compared with rVV-carrying cytokine genes (p < 0.05). These results suggest that attenuated recombinant strains of VV at low doses may potentially be efficient vectors for cancer immunotherapy.