The activity of neurons in the ventromedial hypothalamus (VMH) important for initiating compensatory responses to hypoglycemia is influenced by ambient glucose concentration. In the present study, we used in vivo microdialysis to evaluate interstitial glucose concentrations in rat VMH under various glycemic conditions. Using the zero-net-flux method, steady-state glucose concentration in the VMH was ϳ20% of blood glucose (ϳ1.4 mmol/l) in fed rats but ϳ14% of blood glucose (ϳ0.7 mmol/l) in overnight-fasted rats. During moderate hypoglycemia VMH glucose declined in parallel with blood glucose; however, VMH glucose decreased to a greater degree than blood glucose during a more severe hypoglycemic episode, falling to 10 ؎ 1.2% of blood levels (P < 0.01). To determine whether VMH glucose concentrations were influenced by recurrent episodes of hypoglycemia a second zero-net-flux study was conducted. Steady-state glucose concentrations in the VMH were ϳ20% lower after three episodes of recurrent hypoglycemia, a value 17.8 ؎ 0.8% of blood glucose, although the relative change in VMH glucose levels during the first and fourth hypoglycemic episodes were similar. From these results, we conclude that interstitial glucose concentrations in the VMH are not maintained at a constant level and are more dynamic than previously proposed.