Mitochondrial creatine kinases (MtCK) are members of the creatine kinase (CK) isoenzyme family that are localized in the peripheral intermembrane space and the cristae of mitochondria. Besides the conversion of mitochondrially produced ATP into phosphocreatine and its export as "high energy currency" into the cytosol, additional functions have been proposed for these kinases. More recently, the solved X-ray structures of all four vertebrate CK isoenzymes, as well as mutagenesis, biochemical and biophysical in vitro studies have significantly advanced our understanding of this class of enzymes. This review summarizes in a first part the molecular structure, substrate binding and catalysis of MtCK, as well as genuine MtCK properties like octamer formation or membrane binding, with an emphasis on differences between the two MtCK isoenzymes, sarcomeric and ubiquitous MtCK. A second part describes location and function of MtCKs in their organellar environment, in particular the putative topology of MtCK in proteolipid complexes. Finally, the review discusses the implications of MtCK in human health and disease.