Creatine phosphokinase-mediated transport of energy from the site of production to the site of consumption is a key process for meeting the energy-demands of reactions in cytosol. The mitochondrial creatine phosphokinase (mCPK) plays an important role in this process, with the enzyme activity localized particularly in the mitochondrial contact sites (MiCS). Earlier studies in adult animals have shown that the formation of MiCS varies in response to the energy demand and the physiological state of the heart, and it is stimulated by an increase in [Ca2+]i. However, there is little known about MiCS formation in juvenile hearts, characterized by metabolism different from adult hearts. In the present study we investigated the modulation of MiCS formation via Ca2+ in hearts of 14-day-old rats. The moderate response of MiCS to various stimuli (elevated extracellular Ca2+, diltiazem, cardiac arrest by Cd2+) may refer to a still increased intracellular Ca2+ concentration, the incomplete development of mitochondrial energy production as well as to persistingly high energy demand of the developing heart.