The aim of this study was to evaluate the influence of perfusion media with different glucose concentrations on dialysate levels of lactate, pyruvate, aspartate (Asp), and glutamate (Glu) under basal and hypoxic conditions in rat brain neocortex. Intracerebral microdialysis was performed with the rat under general anesthesia using bilateral probes (o.d. 0.3 mm; membrane length, 2 mm) perfused with artificial CSF containing 0.0 and 3.0 mM glucose, respectively. Basal dialysate levels were obtained 2 h after probe implantation in artificially ventilated animals. Dialysate levels of glucose were also measured for the two different perfusion fluids. The mean absolute extracellular concentration of glucose was estimated by a modification of the no-net-flux method to be 3.3 mmol/L, corresponding to an average in vivo recovery of 6% for glucose. Hypoxia was induced by lowering the inspired oxygen concentration to 3%. Hypoxia caused a disturbance of cortical electrical activity, evidenced by slower frequency and lower amplitudes on the electroencephalogram compared with prehypoxic conditions. This was associated with significant elevations of lactate, Asp, and Glu levels. There were no statistically significant difference in dialysate metabolite levels between the two perfusion fluids, during either normal or hypoxic conditions. We conclude that microdialysis with glucose-free perfusion fluid does not drain brain extracellular glucose in anesthetized rats to the extent that the dialysate lactate, pyruvate, Asp, and Glu levels during basal or hypoxic conditions are altered.