Glucokinase plays a fundamental role in glucose homeostasis by catalysing the high Km phosphorylation of glucose in hepatocytes and islet beta cells. In the brain, the hypothalamus is involved in modulating feeding behaviour through its ability to detect changes in circulating glucose. In this area, the expression of the glucose transporter GLUT2, glucokinase (GK), and ATP-sensitive K+ channels, molecules involved in glucose sensing mechanisms, has been demonstrated. Different studies have suggested that GK is expresed in the hypothalamus; however, it has not been clearly determined if glial and/or neuron cells express this protein (Jetton et al. 1994; Roncero et al. 2000). The neurons in the hypothalamus are in close contact with highly elongated ependymal cells, namely tanycytes. In a previous report, we showed that tanycytes are the main glial cells in the periventricular zone of the hypothalamus and they are expressed GLUT2 (Garcia et al. 2003). In these studies, we analysed the GK expression in hypothalamus tissue. The animals used for the slices were humanely killed. Additionally, we used RT-PCR to demonstrate that mRNAs GK is expressed in liver, islet beta cells and hypothalamic tissues. Real time PCR showed that GK expression in the hypothalamus increased gradually from postnatal day 1 to adult rats. Immunocytochemistry indicates that GK is localized principally in tanycytes that are in direct contact with the cerebrospinal fluid and hypothalamic neurons involved in the glucose sensing mechanism. Primary cultures of hypothalamic tanycytes were used to confirm the gene and protein expression of GK. Immunocytochemical studies showed nuclear and cytoplasmic localization of GK cultured tanycytes. Real time PCR demonstrated an upregulation of GK mRNAs when the cells were incubated with 15mM glucose for 12 h. Based on this evidence, we propose that tanycytes are responsible, at least in part, for a mechanism that allows the hypothalamus to detect changes in glucose concentrations.