The purpose of this study was to investigate the mechanisms explaining improved insulin-stimulated glucose uptake after exercise training in human skeletal muscle. Eight healthy men performed 3 weeks of one-legged knee extensor endurance exercise training. Fifteen hours after the last exercise bout, insulin-stimulated glucose uptake was approximately 60% higher (P < 0.01) in the trained compared with the untrained leg during a hyperinsulinemic-euglycemic clamp. Muscle biopsies were obtained before and after training as well as after 10 and 120 min of insulin stimulation in both legs. Protein content of Akt1/2 (55 +/- 17%, P < 0.05), AS160 (25 +/- 8%, P = 0.08), GLUT4 (52 +/- 19%, P < 0.001), hexokinase 2 (HK2) (197 +/- 40%, P < 0.001), and insulin-responsive aminopeptidase (65 +/- 15%, P < 0.001) increased in muscle in response to training. During hyperinsulinemia, activities of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase (PI3-K) (P < 0.005), Akt1 (P < 0.05), Akt2 (P < 0.005), and glycogen synthase (GS) (percent I-form, P < 0.05) increased similarly in both trained and untrained muscle, consistent with increased phosphorylation of Akt Thr(308), Akt Ser(473), AS160, glycogen synthase kinase (GSK)-3alpha Ser(21), and GSK-3beta Ser(9) and decreased phosphorylation of GS site 3a+b (all P < 0.005). Interestingly, training improved insulin action on thigh blood flow, and, furthermore, in both basal and insulin-stimulated muscle tissue, activities of Akt1 and GS and phosphorylation of AS160 increased with training (all P < 0.05). In contrast, training reduced IRS-1-associated PI3-K activity (P < 0.05) in both basal and insulin-stimulated muscle tissue. Our findings do not support generally improved insulin signaling after endurance training; rather it seems that improved insulin-stimulated glucose uptake may result from hemodynamic adaptations as well as increased cellular protein content of individual insulin signaling components and molecules involved in glucose transport and metabolism.