A single bout of exercise increases insulin sensitivity for several hours after exercise. This is due mainly to increased insulin sensitivity of the muscles involved in the exercise. Although described first in rats 25 years ago the underlying molecular mechanisms remain largely unresolved. Previously we have shown in humans that insulin signalling along the classical IRS1-associated PI3-kinase signalling cascade is not augmented in insulin stimulated muscle after exercise. To further study the effects of prior exercise on other components of the insulin signalling cascade 12 young healthy men performed one-legged dynamic exercise for one hour. Four hours later, insulin-induced glucose uptake was ~ 80% higher (P < 0.05) in previously exercised muscle, measured during a euglycemic-hyperinsulinemic clamp (100mU/ml). Insulin increased (P < 0.05) both IRS-1 and IRS-2 associated PI-3 kinase activity and led to increased (P < 0.001) phosphorylation of Akt on Ser473 and Thr308 in skeletal muscle. Interestingly, in response to prior exercise the level of IRS-2 associated PI-3 kinase activity was higher (P < 0.05) both at basal and during insulin stimulation. The atypical PKC? (aPKC) was similarly activated by insulin in rested and exercised muscle, without detectable changes in subcellular localization or aPKC Thr410 phosphorylation. However, when adding phosphatidylinositol-3,4,5-triphosphate (PIP3), the signalling product of PI-3 kinase, to muscle homogenates, aPKC was more potently activated (P = 0.01) in previously exercised muscle. These findings suggest that exercise increases IRS-2 associated PI-3 kinase activity, and thus the potential for PIP3 production. Furthermore, exercise increases the responsiveness of aPKC to PIP3. These novel molecular sites of interaction between exercise and insulin signalling may be involved in increasing sensitivity of human skeletal muscle to insulin in response to exercise.