Methylphenidate (MPH) is among the main drugs prescribed to treat patients with attention-deficit and hyperactivity disease (ADHD). MPH blocks both the norepinephrine and dopamine reuptake transporters (NET and DAT, respectively). Our study was aimed at further understanding the mechanisms by which MPH could modulate neurotransmitter efflux, using ex vivo radiolabelled neurotransmitter assays isolated from rats. Here, we observed significant dopamine and norepinephrine efflux from the prefrontal cortex (PFC) after MPH (100 µM) exposure. Efflux was mediated by both dopamine and norepinephrine terminals. In the striatum, MPH (100 µM) triggered dopamine efflux through both sodium- and vesicular-dependent mechanisms. Chronic MPH exposure (4 mg/kg/day/animal, voluntary oral intake) for 15 days, followed by a 28-day washout period, increased the firing rate of PFC pyramidal neurons, assessed by in vivo extracellular single-cell electrophysiological recordings, without altering the responses to locally applied NMDA, via micro-iontophoresis. Furthermore, chronic MPH treatment resulted in decreased efficiency of extracellular dopamine to modulate NMDA-induced firing activities of medium spiny neurons in the striatum, together with lower MPH-induced (100 µM) dopamine outflow, suggesting desensitization to both dopamine and MPH in striatal regions. These results indicate that MPH can modulate neurotransmitter efflux in brain regions enriched with dopamine and/or norepinephrine terminals. Further, long-lasting alterations of striatal and prefrontal neurotransmission were observed, even after extensive washout periods. Further studies will be needed to understand the clinical implications of these findings.