Glucose-6-phosphate was measured in microdissected pancreatic islets obtained from obese-hyperglycemic mice. Exposure of the islets in a low glucose medium to the hypoglycemic sulfonylurea compound glibenclamide resulted in a significant decrease of glucose-6-phosphate. This effect might be due to uncoupling of the oxidative phosphorylation leading to an accelerated glycolytic rate. Under the same experimental conditions another stimulator of insulin release, dibutyryl cyclic 3,5-AMP, induced a slight increase in glucose-6-phosphate. Inhibition of the glucose-induced insulin release by epinephrine, diazoxide or omission of Ca+ + resulted in considerably elevated islet levels of glucose-6-phosphate. A reduced availability of Ca+ + may be a common denominator forthis effect on glucose-6-phosphate.