We are studying the enzymes of 2,3-diphosphoglycerate (2,3DPG) metabolism and the regulation of the level of 2,3-DPG in the human red blood cell. 2,3-DPG and other phosphorylated compounds are bound to hemoglobin, with higher affinity for deoxyhemoglobin. We have recently completed a study of the binding constants of 2,3-DPG, ATP, and other metabolically important compounds to hemoglobin. We have also made detailed studies of the factors affecting the rates of the purified enzymes involved in 2,3-DPG metabolism. These results are being used to interpret data obtained from whole cell experiments or in vivo. We have shown that phosphoglycerate mutase is phosphorylated by its cofactor, 2,3-DPG, to form a stable phosphoenzyme containing phosphohistidine. We are studying the amino acid sequence around the phosphorylated histidine residue. We are also studying the kinetics of phosphoryl transfer from the phosphoenzyme.