Recently, numerous studies suggested that in vivo proton magnetic resonance spectroscopy (¹H MRS), with a capability of estimating the concentration of metabolites in brain, could be an appropriate method for investigating schizophrenia (SZ) (1). Previously, the results of MRS indicated that the animal models of SZ represent increased glutamatergic activity on the prefrontal cortex (PFC) which is consistent with the results of never-treated first-episode patients (2). However, owing to spectral overlapping and the J-coupling effect, an accurate quantification of glutamate (Glu) and glutamine (Gln) at the clinical magnetic field , which is necessary for investigating glutamatergic activity, was considered challenging (3). Using high magnetic field and short echo time (TE), the Glu and Gln could be quantified accurately with a small overlap. Therefore, the goal of this study was to investigate the change in the glutamatergic activity on the PFC of the SZ rat model by estimating the changes in the concentration of Glu and Gln with small overlap by using short TE ¹H MRS at 9.4T.Six 8-week-old Sprague–Dawley rats underwent magnetic resonance imaging (MRI) and ¹H MRS. One day after the acquisition of the baseline spectra, all the rats received Mk-801 (0.5 mg/kg) for 6 days. On the sixth day, ¹H MRS was performed 20 min after the administration of Mk-801. T2-weighted images (T2WI) and ¹H MRS were acquired using a 9.4 T Agilent MR scanner. T2WI were acquired in rat brain (slice thickness: 1.5 mm, matrix: 256 × 256) with following parameters TR/Effective TE: 4000/32.95 ms, to locate voxel position. In vivo ¹H MRS were acquired in the voxel (1.5 × 5 × 3 mm³) containing PFC using point-resolved spectroscopy (PRESS) sequence (TR/TE: 5000/13.4 ms, average: 256). Water signal in the voxel was suppressed by variable pulse power and optimized relaxation delays (VAPOR). Unsuppressed water signal was also obtained in the same condition (average: 8). Obtained raw data were processed using linear combination of model spectra (LCModel, version 6.3, Stephen W. Provencher) software to estimate the concentration of the following 17 metabolites: alanine (Ala), aspartate (Asp), creatine (Cr), phosphocreatine (PCr), γ-aminobutyric acid (GABA), glucose (Glc), Gln, Glu, glycerophosphocholine (GPC), phosphocholine (PCh), glutathione (GSH), myo-inositol (mIns), lactate (Lac), N-acetyl aspartate (NAA), N-acetyl-aspartyl-glutamate (NAAG), scyllo-inositol, and taurine (Tau); and total creatine (tCr) was defined to Cr + PCr. Reliability of the estimated concentration was assessed by the Cramer-Raw low bound (CRLB) values; for the accurate interpretation, the metabolites with CRLB value over 20% were excluded. Difference in metabolic concentrations between the two time points was statistically analyzed using a paired-sample t test with PASW statistics 18 (SPSS Inc.) software.Fig. 2 shows the concentrations of the target metabolites (NAA, tCho [GPC+PCho], Cr, GABA, Glu, Gln, Glx, Gln/Glu ratio) acquired from the PFC of rats at baseline and on day 6. The result of the paired-sample t test showed that the concentration of the Glu increased significantly (**p < 0.001) from baseline (13.49 ± 0.99) to that on day 6 (14.30 ± 0.91). Moreover, Glx, which can be considered as the total pool of glutamate, also showed a significant (*p < 0.05) increase between the two time points (18.18 ± 0.92 to 18.99 ± 0.91). No other metabolite shows significant change of the concentration in two time points. Although statistically not significant, the concentration of NAA (p = 0.060) and Cr (p = 0.082) changed. A limitation of our study was that the change in the concentration of NAA and Cr was not elucidated owing to the small sample size. Table 1 illustrates the CRLB values of the target metabolites as % mean ± SD and the results of absolute concentration and relative ratio (/tCr) at the two time point as mean ± SD. Considering the low CRLB value of the target metabolites, our estimation of the concentration was reliable.To investigate the effects of SZ in the glutamatergic activity of the PFC of rats, we compared the concentrations of Glu, Gln, Glx, Gln/Glu ratio at the two time points. Based on the increased concentration of Glu and Glx, which is consistent with previous findings (2), we suggest that SZ-induced change in glutamatergic activity can be accurately detected by ¹H MRS. Moreover, owing to high resolution and minimized J-evolution effects by 9.4 T and the short echo time for this study, the results of the change in glutamatergic activity are more significant than those of the previous study (2). Furthermore, we suggest that our findings can contribute to further ¹H MRS studies investigating the efficacy of antipsychotic drugs for SZ.