Hyperthermia-induced cell inactivation, thermotolerance development, and heat shock protein synthesis were characterized in a Chinese hamster cell line and its two derivatives, respectively, deficient in glycolysis and respiration. No difference was found in the intrinsic thermosensitivity of the cells or in their ability to respond to heat by developing thermotolerance and synthesizing the heat shock proteins. The results indicate that the direct thermal inactivation of either respiration or glycolysis is not an obligatory rate-limiting event in hyperthermic cell killing and that thermotolerance and heat shock proteins are not triggered exclusively as a consequence of damages induced directly by heat in energy metabolism nor do they result specifically in an increased thermostability of respiration or glycolysis. It is concluded that if energy metabolism is involved somehow in the induction by heat of cell death, thermotolerance, and heat shock protein synthesis, it is due to a heat-induced alteration that indirectly causes changes in the cellular energy status rather than to a direct interaction of heat with energy production.