Heat shock sensitizes cells to ionizing radiation, cells heated in S phase have increased chromosomal aberrations, and both Hsp27 and Hsp70 translocate to the nucleus following heat shock, suggesting that the nucleus is a site of thermal damage. We show that the nuclear matrix is the most thermolabile nuclear component. The thermal denaturation profile of the nuclear matrix of Chinese hamster lung V79 cells, determined by differential scanning calorimetry (DSC), has at least 2 transitions at T_m = 48°C and 55°C with an onset temperature of approximately 40°C. The heat absorbed during these transitions is 1.5 cal/g protein, which is in the range of enthalpies for protein denaturation. There is a sharp increase in 1-anilinonapthalene-8-sulfonic acid (ANS) fluorescence with T_m = 48°C, indicating increased exposure of hydrophobic residues at this transition. The T_m = 48°C transition has a similar T_m to those predicted for the critical targets for heat-induced clonogenic killing (T_m = 46°C) and thermal radiosensitization (T_m = 47°C), suggesting that denaturation of nuclear matrix proteins with T_m = 48°C contribute to these forms of nuclear damage. Following heating at 43°C for 2 hours, Hsc70 binds to isolated nuclear matrices and isolated nuclei, probably because of the increased exposure of hydrophobic domains. In addition, approximately 25% of exogenous citrate synthase also binds, indicating a general increase in aggregation of proteins onto the nuclear matrix. We propose that this is the mechanism for increased association of nuclear proteins with the nuclear matrix observed in nuclei isolated from heat-shocked cells and is a form of indirect thermal damage.