NBC Na+/HCO3- cotransporter and NHE1 Na+/H+ exchanger have been associated with cardiac disorders and recently located in mitochondria of coronary endothelial cells (CEC) and cardiomyocytes, respectively. Mitochondrial NHE1 blockade delays mitochondrial permeability transition pore (MPTP) opening and reduces mitochondrial-derived superoxide production, two critical events exacerbated in cells of diseased hearts. Conversely, activation of the NBC isoform, NBCn1, prevented apoptosis in CEC subjected to ischemic stress. We characterized the role of these transporters in mitochondria of hearts from adult spontaneously hypertensive (SHR) and control (Wistar) rats. Expression of NHE1 was analyzed in left ventricular mitochondrial lysates, by immunoblots. NHE1 expression increased by ~40% in hypertrophic SHR compared to control (P<0.05, n=4). To determine if increased expression of NHE1 in cardiac hypertrophy correlates with increase transport activity, mitochondria were loaded with BCECF-AM dye and the maximal rate of pHm change measured after addition of 50 mM NaCl. SHR mitochondria had greater changes in pHm compared to Wistar rats, 0.10±0.01 vs. 0.06±0.01, respectively (P<0.05, n=5). Additionally, mitochondrial suspensions from SHR and control myocardium were exposed to 200 mM CaCl2 to induce MPTP opening (light scattering decrease, LSD) with the consequent mitochondrial swelling (MS). Surprisingly, SHR rats showed smaller LSD and a reduction in MS, 67±10% (n=26), compared to control, 100±8% (n=23). Selective NBC inhibition with 1 mM of the high-affinity S0859 compound, significantly increased MS in both, control 139±10% (n=7), and SHR 115±10% (n=7) mitochondria. Finally, NBCn1 Na+/HCO3- cotransporter increased by twofold its expression in SHR heart muscle mitochondria, compared to normal (P<0.05, n=5). Together our data suggest that increased NBCn1 activity plays a compensatory role in hypertrophic hearts, protecting mitochondria from Ca2+-induced MPTP opening and MS.