Normal and transformed chick embryo cells and their respective ethidium bromide-treated derivatives that are devoid of a functional respiratory chain were comparatively evaluated for their responses to hyperthermia treatment. No significant difference was found between the control and the respiration-deficient cells. The cells have a similar intrinsic thermosensitivity as judged by their capacity to form colonies after treatment at supraoptimal temperatures, and heat triggers in both cases an equal production of heat shock proteins and a strong induction of thermotolerance. In addition, sodium arsenite, carbonyl cyanide m-chlorophenylhydrazone, oligomycin, and antimycin A induce a similar heat shock protein response in the control and the treated cells. Based on these results, it is concluded that the inhibition by heat of the mitochondrial energy production does not constitute an obligatory rate-limiting event in hyperthermic cell killing and that the intracellular signal triggering development of thermotolerance or heat shock protein production does not obligatorily originate from damages to the respiratory chain. Moreover, the results indicate that the modifications responsible for the increased heat resistance of thermotolerant cells may not, or do not necessarily, involve a stabilization of the mitochondrial energy production.