Heat shock proteins (HSPs) are recognized as significant participants in immune reactions. We previously reported that expression of HSP70 in response to hyperthermia, produced using our original magnetite cationic liposomes (MCLs), induces antitumor immunity. In the present study, we examine whether the antitumor immunity induced by hyperthermia is enhanced by hsp70 gene transfer. A human hsp70 gene mediated by cationic liposomes was injected into a B16 melanoma nodule in C57BL/6 mice in situ. At 24 hours after the injection of the hsp70 gene, MCLs were injected into melanoma nodules in C57BL/6 mice, which were subjected to an alternating magnetic field for 30 minutes. The temperature at the tumor reached 43 degrees C and was maintained by controlling the magnetic field intensity. The combined treatment strongly arrested tumor growth over a 30-day period, and complete regression of tumors was observed in 30% (3/10) of mice. Systemic antitumor immunity was induced in the cured mice. This study demonstrates that this novel therapeutic strategy combining the use of hsp70 gene therapy and hyperthermia using MCLs may be applicable to patients with advanced malignancies.