Respiration of Chinese hamster lung V79 cells, as assayed by O2 consumption, increases linearly from 8 to 40°C when plotted in the Arrhenius fashion but is strongly inhibited above 40°C. The protein of mitochondria isolated from V79 cells undergoes structural transitions at 28 and 40°C. This is supported by changes in the fluorescence excitation spectrum of conjugated pyrene maleimide and, to a lesser extent, intrinsic protein fluorophores. Electron spin resonance labelling studies with a derivative of tempo maleimide imply that extensive protein unfolding coincides with the 40°C transition. The structural transition at 40°C correlates well with inhibition of O2 consumption, is irreversible and is probably due to protein denaturation, while the change at 28°C is reversible and has no effect on O2 consumption. Previous studies indicate the presence of a broad lipid transition extending from approximately 8 to 30°C in mitochondrial membranes with all lipids being in the liquid-crystalline state above 30°C. Thus, the onset of the lipid transition may induce the observed protein conformational change at 28°C, but inhibition of respiration above 40°C can be explained by protein denaturation alone. The region from 28 to 40°C of stable protein conformation corresponds to the temperature range of V79 cell growth.