A C18 reversed-phase column and isocratic fluorescence HPLC method for the simultaneous detection of glutamate and gamma-aminobutyric acid (GABA) is described. In this article a fast and more efficient method for the extraction of these neurotransmitters in rat brain tissue is also presented. The supernatant was derivatized with o-phthalaldehyde (OPA) and analyzed by HPLC with fluorescence detection. Intraday reproducibility was 97.0% and 96.7% and interday reproducibility was 97.1% and 93.7% for GABA and glutamate, respectively. Recovery assays indicate that the accuracy of the method for GABA is 99.6+/-2.3% and for glutamate is 101.9+/-1.8%. In addition, the time consumed to run a sample is lower than that described by other authors. Mean elution time was 3.10 min and 8.22 min for glutamate and GABA, respectively. Thus, in a total runtime of less than 9 min both neurotransmitters were detected. Moreover, when compared to the current methods, the extraction solution used here allowed a high drawing out of the neurotransmitters, glutamate and GABA, from the hippocampus, thalamus and prefrontal cortex of the rat brain.