Creatine kinase is involved in the integration of high-energy metabolism in various tissues. In this study the tissue-specific distribution of the mitochondrial isoform was investigated, both by electrophoresis of rat tissue extracts, and by ultrastructural localisation of creatine kinase activity. Furthermore, the influence of uncoupling of oxidative phosphorylation on mitochondrial creatine kinase activity associated with intermembrane contacts was investigated by enzyme cytochemistry and morphometric analysis. The results of the cytochemical survey indicate that contact sites are a prerequisite for creatine kinase to demonstrate enzymatic activity. Moreover, the extent of creatine kinase active membrane contacts depends on the metabolic state of the mitochondrion, as shown for heart mitochondria in vivo and in vitro, before and after treatment with dinitrophenol.