AbstractHeat shock factor-1 (HSF1) is a transcription factor that serves as the major temperature-inducible sensor for eukaryotic cells. In most cell types, HSF1 becomes activated to the DNA binding form at 42°C and mediates the classical heat shock response, protecting the cells from subsequent lethal temperatures. We have recently demonstrated that HSF1 is activated at a lower temperature in T lymphocytes than in most other cell types (39°C vs 42°C), within the physiological range of fever. In this study, we show that T cell activation at fever temperatures not only activates HSF1 but induces the up-regulation of the HSF1 protein and the HSF1-regulated protein, HSP70i. T cells from HSF1 knockout mice proliferate normally under optimal conditions but are impaired in proliferation at physiological fever temperatures and low CO2 concentrations, conditions that do not impair wild-type T cells. This defect in proliferation appears to be mediated by a block in the G1/S transition of the cell cycle and is independent of HSP70. Elevated temperature and low CO2 concentrations resulted in a dramatic reduction of the intracellular reactive oxygen species (ROS) levels in both normal and knockout T cells. Wild-type T cells were able to restore ROS levels to normal within 5 h, whereas HSF1−/− T cells were not. These results suggest that the proliferation defect seen in T cells from HSF1−/− mice at fever temperatures was because of dysregulated ROS levels and that HSF1 is important in maintaining ROS homeostasis and cell cycle progression under the stressful conditions encountered during fever.