Rabbit-muscle glycogen contains covalently bound phosphorus, equivalent to 1 phosphate group per 208 glucose residues. This often disputed, minor component was previously thought to represent a phosphomonoester group at C-6 of a glucose residue. Here we show that more than half the phosphorus is present as a phosphodiester, the remainder being monoester. A novel enzyme activity has been found in muscle that can account for the presence of the phosphodiester in glycogen. This is a UDPglucose:glycogen glucose 1 -phosphotransferase that positions glucose 1 -phosphate on C-6 of glucose residues in glycogen, forming a diester. The phosphomonoester groups present may arise by removal of the glucose residue originally transferred as glucose 1-phosphate. © 1993.