The kinetic properties of the cytoplasmic and the mitochondrial iso-enzymes of creatine kinase from striated muscle were studied in vitro and in vivo. The creatine kinase (CK) iso-enzyme family has a multi-faceted role in cellular energy metabolism and is characterized by a complex pattern of tissue-specific expression and subcellular distribution. In mammalian tissues, there is always co-expression of at least two different CK isoforms. As a result, previous studies into the role of CK in energy metabolism have not been able to directly differentiate between the individual CK species. Here, we describe experiments which were directed at achieving this goal. First, we studied the kinetic properties of the muscle-specific cytoplasmic and mitochondrial CK isoforms in purified form under in vitro conditions, using a combination of P-31 NMR and spectrophotometry. Secondly, P-31 NMR measurements of the flux through the CK reaction were carried out on intact skeletal and heart muscle from wild-type mice and from transgenic mice, homozygous for a complete deficiency of the muscle-type cytoplasmic CK isoform. Skeletal muscle and heart were compared because they differ strongly in the relative abundance of the CK isoforms. The present data indicate that the kinetic properties of cytoplasmic and mitochondrial CK are substantially different, both in vitro and in vivo. This finding particularly has implications for the interpretation of in vivo studies with P-31 NMR.