A biosensor for L-glutamic acid (Glu), constructed by immobilisation of L-glutamate oxidase onto lipid-coated 60 µm radius Pt wire with poly(o-phenylenediamine), was used amperometrically in attempts to detect glutamate in brain extracellular fluid (ECF) in the awake, freely moving rat at 1 s intervals. An implanted carbon paste electrode served to monitor simultaneous changes in brain ascorbate (AA). There were spontaneous, parallel changes in both signals, depending on the behavioural state of the animal, giving rise to a significant positive correlation coefficient between the two variables. The application of a mild stressor (10 s tail pinch) caused an immediate increase in both signals. The effects of inhibiting energy metabolism, together with an existing hypothesis linking Glu and AA in the ECF, were used to conclude that AA does not interfere electrochemically with the biosensor in vivo, that there are parallel changes in extracellular Glu and AA concentrations under normal physiological conditions, and that sub-micromolar physiologically related changes in brain Glu could be detected in the ECF several days after implantation of the electrodes.