Using in vivo microdialysis, we measured hippocampal extracellular glucose concentrations in rats while they performed spontaneous alternation tests of spatial working memory in one of two mazes. Extracellular glucose levels in the hippocampus decreased by 32% below baseline during the test period on the more complex maze, but by a maximum of 11% on the less complex maze. Comparable decreases were not observed in samples taken from rats tested on the more complex maze but with probes located near but outside of the hippocampus. Systemic glucose fully blocked any decrease in extracellular glucose and enhanced alternation on the more complex maze. These findings suggest that cognitive activity can deplete extracellular glucose in the hippocampus and that exogenous glucose administration reverses the depletion while enhancing task performance.