To study the upper limit of glycogen storage in human muscle, two healthy male subjects were infused with glucose and insulin for 8 h reaching plasma concentrations of about 21 mM glucose and approximately 2000 microU/ml insulin. Prior to the infusion subjects performed for 1 h one-legged knee-extensor exercise at 75% of their maximum one-legged work capacity in order to lower muscle glycogen stores in one leg. During the 8-h hyperglycemic clamp procedure, glycogen concentrations increased and levelled off at 2- and 5-fold above the pre-infusion levels in the resting and the working leg, respectively. However, the absolute glycogen levels reached in both legs were quite similar, close to 4 g per 100 g wet muscle (about 1000 mumol/g d.w.), independent of prior exercise. Previous studies have shown that glycogen levels, after a bout of glycogen-depleting exercise and subsequent ingestion of a carbohydrate-rich diet for 3 days, can be increased to values around 3-4 g per 100 g wet muscle. It appears that the maximal attainable glycogen concentration in human muscle seems to be close 4 g per 100 g wet muscle. This glycogen level can thus be reached either by a prolonged infusion of supra-physiological concentrations of glucose and insulin or by glycogen-depleting exercise followed by ingestion of a carbohydrate-rich diet.