Creatine kinase (CK) is an abundant enzyme, important for maintenance of high-energy phosphate homeostasis in many tissues including heart. Double-knockout CK (DbKO-CK) mice missing both the muscle (MM) and sarcomeric mitochondrial (ScMit) isoforms of CK have recently been studied. Despite a large change in skeletal muscle function in DbKO-CK mice, there is little functional change in the heart. To investigate whether there are specific changes in cardiac mitochondrial proteins associated with the loss of MM- and ScMit-CK isoforms, we have used difference gel electrophoresis (DIGE) to compare mitochondrial proteins from wild-type and DbKO-CK mice. Mass spectrometry fingerprinting was used to identify 40 spots as known mitochondrial proteins. We have discovered that the loss of MM- and ScMit-CK isoforms did not cause large scale changes in heart mitochondrial proteins. The loss of ScMit-CK was readily detected in the DbKO-CK samples. We have also detected a large decrease in the precursor form of aconitase. Furthermore, two mitochondrial protein differences have been found in the parent mouse strains of the DbKO-CK mice.