To understand the physiological role of the creatine kinase-phosphocreatine (CK-PCr) system in muscle bioenergetics, a null mutation of the muscle CK (M-CK) gene was introduced into the germline of mice. Mutant mice show no alterations in absolute muscle force, but lack the ability to perform burst activity. Their fast-twitch fibers have an increased intermyofibrillar mitochondrial volume and an increased glycogenolytic/glycolytic potential. PCr and ATP levels are normal in resting M-CK-deficient muscles, but rates of high energy phosphate exchange between PCr and ATP are at least 20-fold reduced. Strikingly, PCr levels decline normally during muscle exercise, suggesting that M-CK-mediated conversion is not the only route for PCr utilization in active muscle.