Introduction: This study conducted spectroscopic analyses using proton (1H) Magnetic Resonance Spectroscopy (at 4 Tesla) in a sample of adolescents with Attention Deficit Hyperactivity Disorder (ADHD), before and after treatment with extended release methylphenidate (OROS MPH), as compared to a sample of healthy comparators. Aims: The main aim of this study is to use 1H MRS to measure differences in brain biochemistry between adolescents with and without ADHD, and to assess changes in cerebral biochemistry, before and after stimulant treatment in ADHD youth. Results: Subjects with ADHD were medically healthy adolescents treated in an open label fashion with OROS MPH (mean dose: 54 mg/day; 0.90 mg/kg/day). Subjects with ADHD were scanned before and after OROS MPH treatment. Healthy comparators were scanned once. Magnetic resonance (MR) spectroscopy studies were performed on a 4.0 T Varian Unity/Inova MR scanner; proton spectra were acquired from the Anterior Cingulate Cortex (ACC). Data were analyzed using MANOVA and repeated measurement ANOVA. Higher metabolite ratios (Glutamate/myo-inositol, Glutamine/myo-inositol, Glutamate + Glutamine/myo-inositol) were observed in the ACC in untreated ADHD subjects as compared to controls, and to treated ADHD youth; these group differences did not reach the a priori threshold for statistical significance. Conclusions: These preliminary findings suggest the presence of glutamatergic abnormalities in adolescents with ADHD, which may normalize with MPH treatment. Larger sample, controlled studies are needed to confirm these preliminary findings.