Yeast and mammalian cytochrome c oxidase activity is inhibited by thiophosphate. This inhibition was observed when using either whole mitochondria or the isolated or reconstituted enzyme. The kinetics of the reduction reaction enabled us to demonstrate that thiophosphate acted on the electron transfer between hemes a and a3. With whole mitochondria, phosphate alone stimulated respiration. The inhibition induced by thiophosphate was suppressed by phosphate only in mitochondria, but not when the isolated enzyme was used. The possibility of a kinetic regulation is discussed.