Substitution of physiologically present macromolecules during isolation of mitochondria and investigation of their functions led to a significant change in regulation of oxidative phosphorylation. The differences compared to conventionally isolated mitochondria were that stimulation of oxidative phosphorylation appeared to rather depend on the activity of peripheral kinases than on the addition of free ADP. The localisation of peripheral kinases such as hexokinase and mitochondrial creatine kinase are described as well as the effects of macromolecules on the regulation of bound hexokinase and of oxidative phosphorylation via this enzyme.