Creatine kinase isoenzymes (CK; EC 2.7.3.2) play a pivotal role in high-energy phosphoryl metabolism through subcellular compartmentation of the creatine-phosphate < = > ATP conversion reaction. In mouse, protein subunits constituting the ubiquitous mitochondrial CK (UbCKmit) and cytosolic B-CK isoforms are co-expressed in various cells and tissues with high and fluctuating energy demands such as brain, retina, smooth muscle, uterus, placenta and spermatozoa. Using targeted mutagenesis via homologous recombination in embryonic stem cells, we have generated mice that are deficient in UbCKmit subunits. These mice are viable and show no overt physical or behavioural abnormalities. Matings between UbCKmit-deficient mice produced normal numbers of offspring, showing that both females and males are completely fertile. Motility patterns of isolated spermatozoa were analyzed and found not to be impaired by absence of UbCKmit. From these results we conclude that UbCKmit is not essential for mouse viability, fertility, maintenance of pregnancy, or delivery.