The role of neutrophils in the anti-tumor effects of hyperthermia was investigated in an experimental rat model, and the efficacy of hyperthermia combined with recombinant human granulocyte colony-stimulating factor (G-CSF) was similarly investigated. AH109A carcinoma cells were transplanted into the hind legs of Donryu rats, then heated by a radio-frequency dielectric heater. In this study, because the myeloperoxidase (MPO) activity of neutrophils was not affected by heating or G-CSF, MPO activity was measured as an index of neutrophil migration into tumor tissue. After hyperthermia, MPO activity in tumor tissue increased significantly, suggesting migration of neutrophils into tumor tissue. Depletion of circulating neutrophils by the intraperitoneal injection of anti-rat neutrophil antibody decreased the anti-tumor effects of hyperthermia. Subsequently, we used hyperthermia plus intraarterial G-CSF to enhance the anti-tumor effect. Hyperthermia was induced 1 h after injection of G-CSF, a time when MPO activity in tumor tissue was maximal. A satisfactory thermal effect was noted even in cases where tissue could not be heated sufficiently. In conclusion, neutrophils have an important role in the anti-tumor effects of hyperthermia, and administration of G-CSF enhances these effects.