Fructose 2,6-bisphosphate has been studied during hypoglycemia induced by insulin administration (40 IU/kg). No changes in content of cerebral fructose 2,6-bisphosphate were found in mild hypoglycemia, but the level of this compound was markedly decreased in hypoglycemic coma and recovered after 30 min of glucose administration. To correlate a possible modification of the concentration of the metabolite with selective regional damage occurring during hypoglycemic coma, we have analyzed four cerebral areas (cortex, striatum, cerebellum, and hippocampus). Fructose 2,6-bisphosphate concentrations were similar in the four areas analyzed; severe hypoglycemia decreased levels of the metabolite to the same extent in all the brain areas studied. The decrease in content of fructose 2,6-bisphosphate was not always accompanied by a parallel decrease in ATP levels, a result suggesting that the low levels of the bisphosphorylated metabolite during hypoglycemic coma could be due to the decreased 6-phosphofructo-2-kinase activity, mainly as a consequence of the fall in concentration of its substrate (fructose 6-phosphate). These results suggest that fructose 2,6-bisphosphate could play a permissive role in cerebral tissue, maintaining activation of 6-phosphofructo-1-kinase and glycolysis.