Mild temperatures such as 40 degrees C are physiological and occur during fevers. This study determines whether mild thermotolerance induced at 40 degrees C can protect HeLa cells against activation of the death receptor pathway of apoptosis by lethal hyperthermia (42-45 degrees C). Protein expression of heat shock proteins (Hsps) 27, 32, 60, 72, 90, and 110 was increased in thermotolerant cells (3 h, 40 degrees C). Lethal hyperthermia (42-43 degrees C) caused cell death by apoptosis, but at 45 degrees C there was a switch to necrosis. Mild thermotolerance protected cells against heat-induced apoptosis (Annexin V labelling). Hyperthermia induced apoptosis through generation of reactive oxygen species (ROS) and death receptor signalling. The antioxidant polyethylene glycol-catalase abrogated increased expression of Fas death ligand and caspase-8 activation in response to lethal hyperthermia (42-43 degrees C). Mild thermotolerance attenuated the heat induction of ROS and FasL, which were initiating events in death receptor activation and signalling. Mild thermotolerance inhibited early events in hyperthermia-induced death receptor apoptosis such as Fas-associated death domain (FADD) translocation to membranes, caspase-8 activation, and tBid translocation to mitochondria. Downstream events in apoptosis such as caspase-3 activation, cleavage of PARP and ICAD, and chromatin condensation were also diminished in thermotolerant cells. It is important to improve knowledge about adaptive responses induced by exposure to mild stresses, such as fever temperatures, which can protect cells against subsequent exposure to lethal stress.