Respiration of Chinese hamster lung V79 cells, as assayed by O2 consumption, increases linearly from 8 to 40 degrees C when plotted in the Arrhenius fashion but is strongly inhibited above 40 degrees C. The protein of mitochondria isolated from V79 cells undergoes structural transitions at 28 and 40 degrees 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 degrees C transition. The structural transition at 40 degrees C correlates well with inhibition of O2 consumption, is irreversible and is probably due to protein denaturation, while the change at 28 degrees 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 degrees C in mitochondrial membranes with all lipids being in the liquid-crystalline state above 30 degrees C. Thus, the onset of the lipid transition may induce the observed protein conformational change at 28 degrees C, but inhibition of respiration above 40 degrees C can be explained by protein denaturation alone. The region from 28 to 40 degrees C of stable protein conformation corresponds to the temperature range of V79 cell growth.