Magnetite cationic liposomes (MCLs) have a positive surface charge and are used as a heating mediator for hyperthermia, because they generate heat in an alternating magnetic field (AMF) due to hysteresis loss. In our previous paper, hyperthermia using MCLs was applied to animals having several types of tumors in mice, rats, hamsters, and rabbits, and a strong anticancer effect was observed. For mice, complete tumor regression was observed when the tumor size was smaller than 5 mm. However, a protocol for large tumors is necessary for the clinical application. In the present paper, a protocol for tumors with a size greater than 15 mm in mice was investigated. MCLs were injected into an MM46 tumor (size, 15 mm) in C3H/HeN mice, which were subjected to AMF for 30 min. The temperature at the surface of the tumor reached 45degreesC and was maintained by controlling the magnetic field intensity. Hyperthermia treatment was repeated twice with 24-h intervals (repeated hyperthermia; RH), and RH was carried out until complete tumor regression was observed. Complete tumor regression was achieved in all mice treated once, twice or six times with RH, and mice acquired antitumor immunity. This protocol, which is termed frequent RH, is a potent approach for cancer therapy.