The cellular location of glucokinase (GK), a key component of the glucose-sensing mechanism of the pancreatic islet, was determined using immunocytochemical techniques. In rat islets, GK immunoreactivity was detected only in beta cells with no immunoreactivity detected in alpha, delta, or pancreatic polypeptide-containing (PP) cells. However, within various beta cells, GK immunoreactivity varied considerably. Most beta cells displayed relatively low levels of cytoplasmic immunoreactivity whereas other beta cells stained intensely for this enzyme. Colocalization studies of GK and GLUT2, the high Km glucose transporter of beta cells, confirmed that these proteins are located in different subcellular domains of beta cells. The lack of GK immunoreactivity in glucagon- and somatostatin-secreting cells in islets suggests that these cells are not directly responsive to glucose or utilize a fundamentally different mechanism for sensing glucose fluctuations. Moreover, the differential expression of GK among pancreatic beta cells suggests that glucose phosphorylation is the probable enzymatic control point for the functional diversity of these cells.