The bystander-killing effect and cyclic induction of tumor necrosis factor (TNF)-alpha gene expression under the heat-inducible promoter gadd 153 (growth arrest and DNA damage) were investigated. The plasmid harboring the TNF-alpha gene under the control of the gadd 153 promoter (pGadTNF) was introduced into cells of the human glioma cell line U251-SP. When the transfected cells were exposed to heat treatment, TNF-alpha gene expression was induced. The heated pGadTNF-transfected cells were co-cultured the nonheated pGadTNF-transfected cells as bystander cells. As a result, TNF-alpha gene expression in the bystander cells was observed. The level of TNF-alpha gene expression was further enhanced in the co-cultured cells. Due to this cyclic induction, a strong cytotoxic effect on the bystander cells was observed. Based on this bystander-killing effect, it was concluded that the transfection of the TNF-alpha gene under the control of the gadd 153 promoter into tumor cells combined with hyperthermia would be a potent tool for cancer therapy.