Summary 1. Cytochrome c oxidase was titrated, under anaerobic conditions, with NADH and phenazine methosulphate in the presence of chelating agents. Three different effects of chelating agents on the titration were found from which it can be concluded that cyanide and azide prevent the reduction of half the haem a and half the copper, EDTA and salicylaldoxime block the reduction of half the copper, and fluoride prevents the reduction of half the haem a. 2. It is concluded that the ratio of cytochrome a: cytochrome a3 is 1. 3. Two different kinds of functionally active copper atoms are present in equal amounts. One is unreactive towards chelating agents such as cyanide, azide, EDTA and salicylaldoxime, whereas, under the conditions of the titration, the reduction of the other can be prevented. 4. Chemical copper determinations confirm the site of action of metal-combining compounds under the conditions of the titration. 5. The cyanide-sensitive copper contributes about one-half of the difference spectrum (oxidized minus reduced) at 830 mμ, whereas the contribution of the other half is due either to haem a of cytochrome a or to the cyanide-insensitive copper or to both. The haem a of cytochrome a3 does not contribute to the difference spectrum at 830 mμ.