To investigate the relationship between glucose uptake and the content of the insulin regulatable glucose transporter, GLUT4, in skeletal muscle at near physiological insulin concentrations in vivo, we measured the effect of a 3h euglycemic insulin-infusion (40 mU m-2 min-1) on glucose uptake and skeletal muscle GLUT4 content in 10 healthy subjects. We found no correlation (r approximately 0.1) between individual muscle GLUT4 content and insulin-stimulated glucose uptake. Mean GLUT4 content in skeletal muscle was reduced by 19 +/- 6.3% (mean +/- SE, p less than 0.02) after insulin infusion. However, when the same subjects were made insulin resistant by infusion of lipid, as evidenced by a reduction of 16 +/- 7.2% (mean +/- SE, p less than 0.05), in insulin-stimulated glucose uptake, the effect of insulin on GLUT4 content was attenuated and no change in GLUT4 content was observed. Our results show that the total content of skeletal muscle GLUT4 is a poor predictor for in vivo response to near physiological insulin concentrations in healthy human subjects.