Studies in rodents have established that GLUT-4 translocation is the major mechanism by which insulin and exercise increase glucose uptake in skeletal muscle. In contrast, much less is known about the translocation phenomenon in human skeletal muscle. In the current study, nine healthy volunteers were studied on two different days. On one day, biopsies of vastus lateralis muscle were taken before and after a 2-h euglycemic-hyperinsulinemic clamp (0.8 mU. kg(-1). min(-1)). On another day, subjects exercised for 60 min at 70% of maximal oxygen consumption (VO(2 max)), a biopsy was obtained, and the same clamp and biopsy procedure was performed as that during the previous experiment. Compared with insulin treatment alone, glucose infusion rates were significantly increased during the postexercise clamp for the periods 0-30 min, 30-60 min, and 60-90 min, but not during the last 30 min of the clamp. Plasma membrane GLUT-4 content was significantly increased in response to physiological hyperinsulinemia (32% above rest), exercise (35%), and the combination of exercise plus insulin (44%). Phosphorylation of Akt, a putative signaling intermediary for GLUT-4 translocation, was increased in response to insulin (640% above rest), exercise (280%), and exercise plus insulin (1,000%). These data demonstrate that two normal physiological conditions, moderate intensity exercise and physiological hyperinsulinemia approximately 56 microU/ml, cause GLUT-4 translocation and Akt phosphorylation in human skeletal muscle.