W hen pancreatic islets isolated from rats infused for 48-72 h with a hypertonic solution of D-glucose were incubated for two successive periods of 10 min each, in the presence first of 16.7 mM and then 2.8 mM D-[U-14C]glucose, the total output of L-lactic acid during the second incubation was as high as that recorded during the first incubation, while the specific radioactivity of L-lactic acid dramatically decreased during the second incubation. In islets from normoglycemic rats, however, the total output of L-lactic acid decreased and its specific radioactivity modestly increased as the concentration of D-glucose was lowered from 16.7 to 2.8 mM. Such contrasting results indicate that in the glycogen-rich islets isolated from glucose-infused rats, the fall in extracellular D-glucose concentration was not accompanied by a parallel fall in glycolytic flux, the decreased utilization of exogenous D-[U-14C]glucose coinciding with stimulation of glycogenolysis. This unusual metabolic situation also coincided with a transient and paradoxical stimulation of insulin release in response to the decrease in extracellular D-glucose concentration. It is proposed, therefore, that the interference of glycogenolysis with glycolysis in pancreatic islets from glucose-infused rats participates in the paradoxical changes in insulin output which represent a typical feature of B-cell glucotoxicity.