In a first series of experiments a glucose oxidase-based biosensor implanted in the striatum of freely moving rats was used to determine the concentration of brain extracellular (ECF) glucose in two distinct ways. With a modification of the zero-net-flux method, in which different concentrations of glucose are infused through a dialysis probe glued to the biosensor, the concentration at which there was no change in glucose current was calculated by regression analysis; this gave an ECF concentration of 351 ± 16 μM. The concentration calculated from the basal current and the in vitro calibration of the biosensor was not significantly different from this. The basal extracellular glucose concentration determined by either method remained constant over a period of several days. In a second series of experiments rats were implanted in the striatum with a Pt/Ir electrode for measurement of regional cerebral blood flow (rCBF, H 2 clearance technique), a carbon paste electrode for monitoring tissue oxygen, and a glucose biosensor for monitoring extracellular glucose. There was a parallel increase in rCBF and oxygen in response to neuronal activation (5 min tail pinch). During the neuronal activation there was a decrease in ECF glucose which was followed by a slow rise that took 30 min to return to basal levels. Finally, a group of rats implanted with a combined glucose biosensor and dialysis probe were given a 5 min tail pinch while the dialysis probe was perfused with either artificial cerebrospinal fluid (aCSF) alone or aCSF with the addition of the β -adrenoceptor antagonist propranolol. Perfusion with aCSF once again produced an initial reduction in extracellular glucose, which was co- extensive with the period of stimulation, followed by a delayed and long-lasting increase in glucose. Propranolol had no effect on basal levels of glucose but suppressed the delayed increase. These results suggests that extracellular glucose in the brain is not derived directly from the blood vascular system but from some other source which is most likely to be astrocytes