Glutamate (Glu) is the main excitatory neurotransmitter in the brain for which several methods have been developed to measure this compound in extracellular brain fluids. Most of these techniques are based on coupling microdialysis to HPLC and they have a resolution time of about 10 min. Here, we present a different approach to measure Glu with a resolution of about 1 min per microdialysis sample, enabling a better relationship to be established between EEG activity and biochemical changes. This new setup was used to determine the time delay between the tip of the microdialysis probe and the site of sample collection, and was accurate to within seconds. Indeed, the measurement of Glu concentrations was linear. Administration of 4-aminopyridine was used to provoke seizure convulsions and under these conditions, biochemical changes and EEG activity were evaluated. These experimental data support the key role of Glu in the initiation of a seizure convulsion.