The purpose of this study was to determine the factors contributing to the ability of exercise to enhance insulin-stimulated glucose disposal. Sixteen insulin resistant nondiabetic and seven type 2 diabetic subjects underwent two hyperinsulinemic (40 mU/m²/min) clamps, once without and once with concomitant exercise at 70% VO₂peak. Exercise was begun at the start of insulin infusion and was performed for 30 minutes. Muscle biopsies of the vastus lateralis were performed before and after 30 minutes of insulin infusion (immediately after the cessation of exercise). Exercise synergistically increased insulin-stimulated glucose disposal in nondiabetics (from 4.6 ± 0.4 to 9.5 ± 0.8 mg/kg FFM/min) and diabetics (from 4.3 ± 1.0 to 7.9 ± 0.7 mg/kg FFM/min). The rate of glucose disposal also was significantly greater, in each group after cessation of exercise. Exercise enhanced insulin-stimulated increases in glycogen synthase fractional velocity, from 0.07 ± 0.02 to 0.22 ± 0.05 (P < 0.05) in control subjects and from 0.08 ± 0.03 to 0.15 ± 0.03 (P < 0.01) in diabetic subjects. Exercise also enhanced insulin-stimulated glucose storage (glycogen synthesis) in both nondiabetic (2.9 ± 0.9 vs. 4.9 ± 1.1 mg/kg FFM/min) and diabetic (1.7 ± 0.5 vs. 4.2 ± 0.8 mg/kg FFM/min) subjects. Moreover, increased glucose storage accounted for the increase in whole body glucose disposal seen when exercise was performed during insulin stimulation in both group; the effects of exercise to enhance glucose disposal and glucose storage were correlated (r = 0.93, P < 0.001). Exercise synergistically enhanced insulin-stimulated IRS-1 associated PI 3-kinase activity (P < 0.05) and Akt serine 473 phosphorylation (P < 0.05) in nondiabetic subjects, but had little effect in diabetic subjects. The data indicate that exercise, performed in conjunction with insulin infusion, synergistically increases insulin-stimulated glucose disposal, compared to the effect of insulin alone. In nondiabetic and diabetic subjects, increased glycogen synthase activation is likely to be involved, in part, in this effect. In addition, in nondiabetic subjects, but not diabetic subjects, exercise-induced enhancement of insulin-stimulation of the PI 3-kinase pathway is also likely to be involved in the exercise induced synergistic enhancement of glucose disposal.