Pancreatic islets from obese-hyperglycemic mice were used for studying a proposed relationship between insulin release and the β-cell content of 6-phosphogluconate. A short period of ischemia reduced the amount of this intermediate in the islets but not in the exocrine pancreas. There was a steep rise of 6-phosphogluconate in microdissected islets when the extracellular glucose concentration was increased from 0.6 to 3.0 mg/ml. A plateau of about 40 µmoles 6-phosphogluconate per kg islet dry weight was reached at 10 mg/ml of glucose. 6-phosphogluconate remained unaffected when the microdissected islets were exposed to the insulin secretagogues glibenclamide or dibutyryl-3,5-cyclic AMP. After addition of epinephrine to or omission of Ca++ from a high glucose medium, there was, however, a significant elevation of 6-phosphogluconate. The results indicate that the islet content of 6-phosphogluconate does not necessarily reflect the rate of insulin release. 1 Supported by grants from the United States Public Health Service (AM-12535), the Swedish Medical Research Council (12x-562), the Nordic Insulin Fund and the Medical Faculty of Umeå. For generous supplies of material we are indebted to Farbwerke Hoechst AG, Frankfurt/M, Germany (glibenclamide), AB Kabi, Stockholm, Sweden (serum albumin) and Schering Corporation, Bloomfield, NJ. (diazoxide)