On the basis of previous indirect measurements, skeletal muscle has been implicated as the major site of glucose uptake and it has been suggested that muscle glycogen formation is the dominant pathway. However, direct measurements of the rates of glycogen synthesis have not been possible by previous techniques. We have developed 13C NMR methods to measure directly the rate of human muscle glycogen formation from infused, isotopically labeled [1-13C]glucose. We show that under conditions of imposed hyperglycemia and hyperinsulinemia, a majority of the infused glucose was converted to muscle glycogen in a normal man. This directly shows that muscle is the major site of glucose disposal under these conditions, and provides quantitation of the glucose flux to muscle glycogen.