Intratumoral inoculation with a herpes simplex virus (HSV) mutant, G207, as an in situ cancer vaccine has been shown to inhibit tumor growth by inducing tumor-specific immune responses. Here, as a step toward the clinical application of this therapeutic approach, we evaluated different protocols for enhancing the antitumor effect. First, in a bilaterally established tumor model with CT26 colon carcinoma, we demonstrated that multiple intratumoral inoculations with G207 induced a greater antitumor effect on both the inoculated and distant tumors than did 1 or 2 inoculations. Second, to boost this antitumor effect, we developed 2 strategies: multiple in situ cancer vaccines with G207 in combination with systemic administration of recombinant interleukin-12 (rIL-12) (G207/systemic rIL-12) or local administration of rIL-12 (G207/local rIL-12). The antitumor effects in both the inoculated and distant tumors by the combined treatments were significantly greater than by either G207 or rIL-12 treatment alone. G207/systemic rIL-12 and G207/local rIL-12 mediated the complete regression of both the inoculated and distant tumors in 67% and 79% of the animals, respectively. These results indicate that multiple intratumoral inoculations of G207 and systemic or local rIL-12 administration work synergistically to facilitate tumor regression and that this combination of treatments may have potential for treating cancer metastasis.