The antitumor efficacy of human melanoma-associated antigen (hgp100) and chemokine CCL21 in combination with interleukin-12 (IL-12) was evaluated in a syngeneic melanoma mouse model. The rationale for this approach was based on previous studies showing that the efficacy of IL-12 therapy in melanoma patients correlated with the presence of antibodies against tumor-associated antigens. We have previously shown that application of xenogeneic human gp100 DNA (hgp100 DNA) is protective against mouse B16 melanoma. Furthermore, the chemokine CCL21 has the ability to chemoattract both dendritic cells (DCs) and T lymphocytes. We show here that intratumoral injection of IL-12-encoding DNA (IL-12 DNA) in combination with hgp100- encoding DNA (hgp100 DNA) into tumor-bearing mice led to a strong antitumor effect. Coapplication of IL- 12 DNA with CCL21-encoding DNA (CCL21 DNA) or recombinant CCL21 (recCCL21) protein also showed some efficacy. Triple therapy with IL-12 DNA, hgp100 DNA, and CCL21 DNA, however, showed less effect on tumor growth than double therapy with IL-12 DNA and hgp100 DNA. These findings open a new route of investigation of IL-12 and gp100 or other tumor-associated antigens in the immunotherapy of a variety of tumors.