Studies have shown a relationship between energy metabolism and methylphenidate (MPH); however, there are no studies evaluating the effects of MPH in Krebs cycle. So, we investigated if MPH treatment could alter the activity of citrate synthase (CS), malate dehydrogenase (MD), and isocitrate dehydrogenase (ID) in the brain of young and adult Wistar rats. Our results showed that MPH (2 and 10 mg/kg) reduced CS in the striatum and prefrontal cortex (PF), with MPH at all doses in the cerebellum and hippocampus after chronic treatment in young rats. In adult rats the CS was reduced in the cerebellum after acute treatment with MPH at all doses, and after chronic treatment in the PF and cerebellum with MPH (10 mg/kg), and in the hippocampus with MPH (2 and 10 mg/kg). The ID decreased in the hippocampus and striatum with MPH (2 and 10 mg/kg), and in the cortex (10 mg/kg) after acute treatment in young rats. In adult rats acute treatment with MPH (2 and 10 mg/kg) reduced ID in the cerebellum, and with MPH (10 mg/kg) in the cortex; chronic treatment with MPH (10 mg/kg) decreased ID in the PF; with MPH (2 and 10 mg/kg) in the cerebellum, and with MPH at all doses in the hippocampus. The MD did not alter. In conclusion, our results suggest that MPH can alter enzymes of Krebs cycle in brain areas involved with circuits related with attention deficit hyperactivity disorder; however, such effects depend on age of animal and treatment regime.