Purpose: To examine the heat sensitivity of DNA-dependent protein kinase (DNA-PK) activity in a variety of cultured mouse, hamster and human cell lines. Materials and methods: Eight cell lines, which have been routinely used in our laboratory, were examined. Cells were heated at 44.0±0.05°C and DNA-PK activity was measured by a DNA-pull-down assay followed by gel-electrophoresis. Cellular sensitivity to hyperthermia and/or X-ray was evaluated by a colony formation assay. Results: In mouse FSA1233 and FM3A cells, DNA-PK activity dropped to 15-16% of unheated control after 20 min of heating. In Chinese hamster V79 and CHO-K1 cells, kinase activity did not change appreciably after 20 min treatment but decreased to 60-70 and 22-23% after 40 or 60 min treatment, respectively. However, even after 180 min treatment, DNA-PK activity remained almost intact in human MOLT-4, MKN45 and A7 cells, and decreased only slightly in U937 cells. Hyperthermic radiosensitization was seen even in human cells but, as a trend, it was small compared with rodent cells. Conclusions: The heat sensitivity of DNA-PK was clearly different among mouse, hamster and human cells. The results suggested a possibility that the role of DNA-PK inactivation in hyperthermic radiosensitization might be variable, depending on cells, and would reinforce the warning that the direct extrapolation of data from rodent cells might lead to overestimation of the effectiveness of hyperthermia on human cancer.