We have constructed an antibody interleukin-12 (IL-12) fusion protein (mscIL-12.her2.IgG3) that demonstrates significant antitumor activity against the murine carcinoma CT26-expressing human HER2/neu. We now report that this antitumor activity is dose dependent and comparable to or better than recombinant murine IL-12 (rMuIL-12) using subcutaneous and metastatic models of disease. The antitumor activity of mscIL-12.her2.IgG3 is reduced in Rag2 knockout mice, suggesting that T cells play a role in tumor rejection. In SCID-beige mice, the antitumor activity is further reduced, suggesting that natural killer (NK) cells or macrophages or both are also important. The isotype of the antibody response to HER2/neu is consistent with a switch from a Th2 to a Th1 immune response and the infiltration of mononuclear cell in tumors from mice treated with mscIL-12.her2.IgG3. Immunohistochemistry reveals that mscIL-12.her2.IgG3 is antiangiogenic. Thus, the mechanism of the antitumor activity exhibited by mscIL-12.her2.IgG3 is highly complex and involves a combination of T and NK cell activity, a switch to a Th1 immune response, and antiantiogenic activity. This is the first study comparing the in vivo antitumor activity of an antibody-IL-12 fusion protein and free IL-12. Our results suggest that antibody-IL-12 fusion proteins may be useful for the treatment of human cancer.