It is known that mitochondria can accumulate calcium, which regulates energy metabolism and cell death. About 90% of energy of cardiomyocytes is synthesized in mitochondria. Heart cells are also affected by the rapid changes in the Ca2+ concentration in the cytoplasm. Therefore, mitochondrial Ca2+-accumulation ability is crucial. The aim of our work was to study the accumulation of Ca2+ in isolated rat heart mitochondria in the presence of mitochondrial potential and different extramitochondrial Ca2+ concentrations. Isolated organelles were loaded with fluorescent dye Fluo-4 AM (2,5 µmol/l) at a temperature of 26°C for 30 min. It has been revealed that under these conditions high mitochondrial potential was maintained sufficiently, which is necessary for the functioning of the calcium transporting system in organelles. We established that mitochondria have a limited ability to store ionized calcium, as addition of Ca2+ ion in concentrations of 10, 20, 50 µmol/l ensures a certain level of accumulation in organelles with further fluorescent signal growth cessation. Addition of 100 µmol/l Ca2+ to isolated mitochondria resulted in a significant increase in fluorescence intensity (46% in the fifth minute, compared to the fluorescence when 20 µmol/l Ca2+ was added) and likely to activation of cation release. It was shown that ruthenium red (10-5 mol/l), an inhibitor of Ca2+ -uniporter, prevented accumulation of calcium ions in organelles by 89%, in the presence of 100 µmol/l Ca2+ . It was clearly seen that heart mitochondria require Mg2+-ATP complex (3 mmol/l) to accumulate Ca2 +, likely to maintain the inner membrane potential, activity of Ca2+ uniporter and energetic processes in organelles. Thus, the process of Ca2+ accumulation in rat heart mitochondria requires the maintenance of mitochondrial potential, activity of Ca2+-uniporter, depends on extramitochondrial Ca2 +concentration and presence of Mg2+-ATP complex.