Glucose metabolism is of vital importance in normal brain function. Evidence indicates that glycolysis, in addition to production of ATP, plays an important role in maintaining normal synaptic function. In an effort to understand the potential involvement of a glycolytic intermediate(s) in synaptic function, we have prepared [3- 32 P]1,3-bisphosphoglycerate and [ 32 P]3-phosphoglycerate and sought their interaction with a specific nerve-ending protein. We have found that a 29-kDa protein is the major component labeled with either [3- 32 P]1,3-bisphosphoglycerate or [ 32 P]3-phosphoglycerate. The protein was identified as monophosphoglycerate mutase (PGAM). This labeling was remarkably high in the brain and synaptosomal cytosol fraction, consistent with the importance of glycolysis in synaptic function. Of interest, fructose-2,6-bisphosphate (Fru-2,6-P 2 ) inhibited PGAM phosphorylation and enzyme activity. Moreover, Fru-2,6-P 2 potently stimulated release of [ 32 P]phosphate from the 32 P-labeled PGAM (EC 50 = 1 µm), suggesting that apparent reduction of PGAM phosphorylation and enzyme activity by Fru-2,6-P 2 may be due to stimulation of dephosphorylation of PGAM. The significance of these findings is discussed.