Homeostatic control of brain glucose involves the hypothalamus and lower brainstem. We have identified and characterised glucosensing neurons in the vagal complex of the brainstem using in situ hybridisation, single-cell RT-PCR and whole-cell patch-clamp recordings from 200 μm thick brain slices. Slices were obtained from juvenile Sprague-Dawley rats humanely killed following halothane anaesthesia. After 30 min recovery at 34°C slices were kept at 22°C in artificial cerebrospinal fluid (ACSF). Whole-cell recordings from 153 cells were performed under visual control. From 30 cells cytoplasm was aspirated into the electrode after patch-clamp recording and used for single-cell RT-PCR. In situ hybridisation was performed with biotin-labelled riboprobes for glucokinase (GLK) on 30μm coronal brainstem sections cut from perfuse-fixed tissue on a cryostat. Results were visualised with an avidin-alkaline phosphatase conjugate and BCIP/NBT colour substrate. To identify glia, slices were also incubated with an anti-GFAP (glial fibrillary acidic protein) Cy3-conjugate. Hypoglycemic responses of neurons in the dorsal vagal nucleus and the nucleus tractus solitarius were tested by a change from 10mM to 0mM ACSF glucose. Most neurons did not respond to this challenge within 5min (non-responsive: NR). 15 neurons tested depolarised from -59±2 mV within 155±31s (mean±s.e.m) of glucose removal (glucose-inhibited: GI). 11 hyperpolarised by 14±2mV and stopped firing within 53±7s (glucose-excited: GE). The hyperpolarisation was reversed by 10mM glucose or 100μM tolbutamide, a K_ATP channel blocker. Single cell RT-PCR showed that all GI (n=3), 2 of 3 GE, and 0 NR (n=24) expressed GLK. 1 GI, the 2 GLK-positive GE and 11 NR also expressed SUR1, a K_ATP channel subunit. In situ hybridisation revealed widespread staining for GLK across the lower brainstem (n=7). In the vagal complex strongly stained cells were found in the dorsal vagal nucleus, nucleus tractus solitarius and area postrema. Glucokinase was not co-localised with GFAP (n=4), suggesting that glucosensors are primarily neuronal. We have demonstrated that GI and GE neurons exist in the brainstem and suggest that GLK is essential for their function. GE cells work analogous to pancreatic β-cells requiring both GLK and K_ATP channels. Vagal glucosensors might be involved in the regulation of vagal outflow to the pancreas providing CNS influence on blood glucose levels dependent on brain glucose availability.