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The aim of the present in vivo microdialysis study was to investigate the relation between feeding and changes in glucose concentrations in the rat ventromedial hypothalamus (VMH). Absolute ambient glucose concentrations in VMH were 1.43 mm in non-deprived rats as compared to 0.94 mm after 24-h food deprivation. To examine whether feeding influences hypothalamic glucose, changes of glucose concentration over time were determined relative to a baseline. Experiments were conducted in relation to both, nutritional state (food-deprived rats vs. non-deprived rats) and feeding conditions throughout the experiment (freely feeding rats vs. rats without access to food). The results of this microdialysis study show clearly that glucose concentration in the VMH of rats increases significantly in relation to food intake. The data demonstrate that a 24-h food deprivation before the experiment further augments this increase (up to 350% from baseline) as compared to non-deprived conditions (up to 60% from baseline). However, the magnitude of food related increase in VMH glucose does not correlate with the individual amount of food eaten. In conclusion, the present study shows for the first time that VMH glucose concentrations increase with food intake in the early dark phase, indicating that such changes do not only occur after pharmacological treatment, but also under physiological feeding conditions. The results further indicate that the feeding related increase in VMH glucose depends on the nutritional state of the organism.