To understand the role of creatine kinase (CK) in cardiac excitation‐contraction coupling, CK‐deficient mice (CK–/–) were studied in vitro and in vivo. In skinned fibers, the kinetics of caffeine‐induced release of Ca²⁺ was markedly slowed in CK–/– mice with a partial restoration when glycolytic substrates were added. These abnormalities were almost compensated for at the cellular level: the responses of Ca²⁺ transient and cell shortening to an increased pacing rate from 1 Hz to 4 Hz were normal with a normal post‐rest potentiation of shortening. However, the post‐rest potentiation of the Ca²⁺ transient was absent and the cellular contractile response to isoprenaline was decreased in CK–/– mice. In vivo, echocardiographically determined cardiac function was normal at rest but the response to isoprenaline was blunted in CK–/– mice. Previously described compensatory pathways (glycolytic pathway and closer sarcoplasmic reticulum‐mitochondria interactions) allow a quasi‐normal SR function in isolated cells and a normal basal in vivo ventricular function, but are not sufficient to cope with a large and rapid increase in energy demand produced by β‐adrenergic stimulation. This shows the specific role of CK in excitation‐contraction coupling in cardiac muscle that cannot be compensated for by other pathways.