The present study was aimed to investigate the effect of О-carboxymethyl hydroxylamine (O-CMH) on heart function in conditions of Ca2+ loads and mitochondrial permeability transition pore (MPTP) opening in cardiac mitochondria. OCMH (50 mg per kg) was dissolved in physiological solution and injected intraperitoneally 30 min before the experiment. Rat isolated hearts were Langendorf-perfused and subjected to increased concentration of Ca2+ in perfusion solution ranged from 1.7 to 12.5 mmoles/L. The heart function was assessed by measuring the LVDP, dP/dt, the coronary flow, the heart rate. The opening of MPTP was estimated by monitoring Ca2+ induced mitochondria swelling at 520 nm. The results showed that pretreatment with O-CMH significantly depressed the initial contractile activity of the isolated rat hearts and the coronary flow. Further modeling of Ca2+ loads was accompanied with lower increment of LVDP and dP/dt comparing to control rats indicating decreased functional reserves and low effectiveness of Ca2+ management in O-CMH pretreated rats. Additionally, cardiac mitochondria in O-CMH group were more sensitive to Ca2+ showing maximum swelling at 10-5 moles/L in the incubation medium vs 10-4 moles/L in control group (p<0.05). Pre-incubation of cardiac mitochondria with O-CMH in concentration of 10-5, 10-4 and 10-3 moles/L leaded to increased Ca2+ induced swelling by 17.8, 20 and 44% respectively. Thus, we have found that inhibition of mitochondrial H2S synthesis pathway increase sensitivity of cardiac mitochondria to Ca2+ that lead to mitochondrial swelling and decreased ability of myocardium to manage Ca2+ homeostasis by cardiomyocytes in conditions of Ca2+ load. This might be proposed as the physiological role of H2S synthesized in mitochondria by 3-MPST. Key words: .