Although the absence of intracellular (IC) free glucose is direct evidence of glucose transport being the rate-limiting step for muscle glucose disposal at euglycemia, the scarcity of data in humans during hyperglycemia precludes any definitive conclusion. In the present study, 13C and 31P in vivo nuclear magnetic resonance (NMR) data from two separate groups of subjects were combined to measure IC free glucose in the human skeletal muscle. When these noninvasive tools were used with an infusion of [1-13C]glucose, a steady-state concentration of 1.2 +/- 0.2 mmol IC glucose/l IC water was observed at the end of a 2-h hyperglycemic clamp with somatostatin infusion, during which glycemia was maintained at approximately 22 mmol/l and insulinemia at approximately 5 mU/l. Despite this moderate glucose accumulation, the persistence of a large transmembrane glucose gradient suggests that the posttransport steps do not play a significant role in the control of muscle glucose disposal in these specific conditions, relevant to insulinopenic diabetic patients.