Glucokinase (GK) is an essential component of glucose sensing in the liver, pancreas and brain. To investigate the role of distinct populations of glucokinase expressing neurones in hypothalamic nuclei it is necessary to measure levels of GK activity within them under different physiological conditions. Previous methods for direct measurement of GK activity¹ requires lengthy preparation and uses large amounts of tissue so is not suitable for hypothalamic measurements where nuclei can be less than 1mm³. We have modified a previously used assay² for use in individual nuclei of the rat hypothalamus. The GK activity assay measures production of NADPH in the following reaction. GK catalyses the phosphorylation of glucose to glucose-6-phosphate (G-6-P) in the presence of ATP. G-6-P dehydrogenase converts G-6-P to 6-phophate-D-gluconate and generates NADPH. Absorbance by NADPH at 340nm is measured using a spectrophotometer to calculate NADPH produced in the reaction and therefore GK activity. Inhibitors of other hexokinases are added to ensure that only GK can catalyse the reaction. We tested the assay using GK from bacillus stearothermophilus (Sigma Ltd, Poole, Dorset, UK). Increasing GK resulted in a proportional increase in NADPH production. We then measured GK activity in homogenised hypothalamic nuclei of rats which had been microdissected using a punch biopsy. Active GK was measured at 7.13±0.19 U/mg of protein (n=10) in the ventromedial nucleus, an area widely associated with control of glucose homeostasis. Interestingly, GK activity was significantly higher in the nearby arcuate nucleus (9.83±0.42U/mg, p<0.0001, n=11) and slightly lower in the paraventricular nucleus (6.75±0.58U/mg, n=11). In conclusion, this tool offers a direct measure of GK activity in hypothalamic tissue. This can be used to detect changes in GK activity using small amounts of tissue and is useful in the investigation of glucose sensing in the brain.