Exercise has been shown to increase insulin-stimulated skeletal muscle glucose transport. Activation of phosphatidylinostol 3-kinase (PI 3-kinase) is required for insulin to stimulate glucose transport. The present study was designed to investigate whether treadmill running (60 min, 8% grade, 30 m/min) augments insulin-stimulated activation of PI 3-kinase. Insulin dramatically increased insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation (p < 0.05). Treadmill running did not induce IRS-1 tyrosine phosphorylation, not did it alter insulin-stimulated IRS-1 tyrosine phosphorylation. Insulin increased PI 3-kinase activity by 3.0-fold (over basal activity) in white muscle and 5.2-fold in red muscle (p < 0.05). Exercise did not alter basal PI 3-kinase activity in either white or red muscle. However, in response to exercise, insulin-stimulated PI 3-kinase activity was significantly increased in both muscle fibers (p < 0.05). These results suggest that increased insulin responsiveness induced by exercise may be due, in part, to enhanced insulin-stimulated PI 3-kinase activity.