Tissue accumulation of high amounts of D-2-hydroxyglutaric acid (DGA) and l-2-hydroxyglutaric acid (LGA) is the biochemical hallmark of the inherited neurometabolic disorders D-2-hydroxyglutaric aciduria (DHGA) and l-2-hydroxyglutaric aciduria (LHGA), respectively. Patients affected by DHGA predominantly present neurological and cardiomuscular symptoms, while those with LHGA have mainly severe neurological symptoms. Lactic aciduria and/or lactic acidemia may also occur in both disorders, suggesting mitochondrial dysfunction. We have previously reported that cytochrome c oxidase (COX) activity is severely inhibited by DGA in rat cerebral cortex and human skeletal muscle. In the present study, we initially evaluated the role of DGA and LGA on the mitochondrial respiratory chain complex activities, as well as CO2 on production in cardiac and skeletal muscle from 30-day-old Wistar rats. DGA significantly inhibited COX and ATP synthase (F0F1-ATP synthase) activities, in contrast to the other activities of the respiratory chain enzymes which were not affected by DGA in both muscular tissues. In addition, CO2 production was also markedly reduced by DGA in rat skeletal and cardiac muscles. On the other hand, LGA did not interfere with any of the respiratory chain complex activities studied, neither with CO2 generation. We also measured mitochondrial respiratory parameters in rat brain mitochondrial preparations in the presence of DGA and LGA. Both metabolites significantly lowered the respiratory control ratio in the presence of glutamate/malate and succinate. Since the metabolites stimulated oxygen consumption in state IV and compromised ATP formation, it can be presumed that these organic acids might act as endogenous uncouplers of mitochondria respiration. Moreover, COX activity linked to TMPD-ascorbate was significantly reduced by DGA in the brain mitochondrial enriched fractions. Finally, DGA and LGA reduced cell viability of rat cerebral cortex slices, as determined by the MTT assay. In case our in vitro data also occur in vivo, it may be presumed that impairment of energy metabolism may contribute to the understanding of the clinical features mainly of patients affected by DHGA.