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Glucokinase (GK) is a metabolic sensor involved in the regulated release of insulin from pancreatic β-cells. Activators of GK may therefore have a possible role in the therapeutic management of type 2 diabetes. Here, we have examined the actions of the GK activator GKA50 on insulin secretion and intracellular Ca[sup 2+] homeostasis ([Ca[sup 2+]][sub i]) in isolated mouse, rat and human islets of Langerhans, and in the MIN6 insulin-secreting cell line. In rodent islets and MIN6 cells, 1µM GKA50 was found to stimulate insulin secretion and raise [Ca[sup 2+]][sub i] in the presence of glucose (2-10mM). Similar effects on insulin release were also seen in isolated human islets. GKA50 (1µM) caused a leftwards shift in the glucose-concentration response profiles and the EC[sub 50] values for glucose were shifted by 3mM in rat islets and approximately 10mM in MIN6 cells. There was no significant effect of GKA50 on the maximal rates of GSIS. In the absence of glucose GKA50 failed to elevate [Ca[sup 2+]][sub i] (1µM GKA50) or stimulate insulin release (30nM-10µM GKA50). At 5mM glucose the EC[sub 50] for GKA50 in MIN6 cells was approximately 0.3µM. Inhibition of GK with mannoheptulose or 5-thioglucose selectively inhibited the action of GKA50 on insulin release but not the effects of tolbutamide. Similarly, compromising glucose metabolism by 3-methoxyglucose prevented GKA50-induced rises in [Ca[sup 2+]][sub i] but not the actions of tolbutamide. Finally, we also show that the K[sub ATP] channel agonist diazoxide (200µM) inhibited GKA50-induced insulin release and its elevation of [Ca[sup 2+]][sub i]. This study illustrates that GKA50 is a glucose-like activator of βcell metabolism and a Ca[sup 2+]-dependent modulator of insulin secretion. [ABSTRACT FROM AUTHOR]