The influence of protein phosphorylation on the kinetics of cytochrome c oxidase was investigated by applying Western blotting, mass spectrometry and kinetic measurements with an oxygen electrode. The isolated enzyme from bovine heart exhibited serine, threonine and/or tyrosine phosphorylation in various subunits, except subunit I, by using phosphoamino acid specific antibodies. The kinetics revealed slight inhibition of oxygen uptake in the presence of ATP, as compared to the presence of ADP. Mass spectrometry identified the phosphorylation of Ser34 at subunit IV and Ser4 and Thr35 at subunit Va. Incubation of the isolated enzyme with protein kinase A, cAMP and ATP resulted in serine and threonine phosphorylation of subunit I, which was correlated with sigmoidal inhibition kinetics in the presence of ATP. This “allosteric ATP-inhibition” of cytochrome c oxidase was also found in rat heart mitochondria, which have been rapidly prepared in the presence of protein phosphatase inhibitors. The isolated rat heart enzyme, prepared from the mitochondria by blue native gel electrophoresis, showed serine, threonine and tyrosine phosphorylation of subunit I. It is concluded, that the allosteric ATP-inhibition of cytochrome c oxidase, previously suggested to keep the mitochondrial membrane potential and thus the ROS production in cells at low levels, occurs in living cells and is based on phosphorylation of cytochrome c oxidase subunit I.