The enzyme glucokinase controls glucose metabolism in islets and is proposed to be the glucose sensor in pancreatic beta-cells. This concept was developed from studies with rodents and it remained to be explored whether it also applies to man. Studies in man were hampered, however, by the difficulty in obtaining well-preserved pancreatic islet tissue and also because the high activity of hexokinase made it difficult to measure glucokinase. To overcome these obstacles, quantitative histochemical sampling techniques were developed allowing precise dissection of pure human islet tissue and a newly designed radiometric microassay was used, avoiding hexokinase interference, and providing the sensitivity necessary to measure the relatively low glucokinase activity in small samples of tissue obtained from brain-dead tissue donors. The present data indicate that glucokinase is present in human pancreatic islet tissue and is not found in the exocrine pancreas. The enzyme's Vmax with D-glucose as substrate was similar to the Vmax for glucose utilization reported previously for intact, isolated human islets and the enzyme's Km for D-glucose was about 5 mM. Since glucokinase was also present in islet tissue of hamster, mouse, and rat, it is suggested that the glucokinase-glucose sensor concept has general applicability and that it could explain many aspects of the physiology and pathology of glucose homeostasis. This well-defined pancreatic islet glucokinase-glucose sensor should, therefore, be incorporated in any comprehensive model of glucose homeostasis.