Wojtaszewski, Jørgen F. P., Bo F. Hansen, Birgitte Ursø, and Erik A. Richter. Wortmannin inhibits both insulin- and contraction-stimulated glucose uptake and transport in rat skeletal muscle. J. Appl. Physiol. 81(4): 1501–1509, 1996.—The role of phosphatidylinositol (PI) 3-kinase for insulin- and contraction-stimulated muscle glucose transport was investigated in rat skeletal muscle perfused with a cell-free perfusate. The insulin receptor substrate-1-associated PI 3-kinase activity was increased sixfold upon insulin stimulation but was unaffected by contractions. In addition, the insulin-stimulated PI 3-kinase activity and muscle glucose uptake and transport in individual muscles were dose-dependently inhibited by wortmannin with one-half maximal inhibition values of ∼10 nM and total inhibition at 1 μM. This concentration of wortmannin also decreased the contraction-stimulated glucose transport and uptake by ∼30–70% without confounding effects on contractility or on muscle ATP and phosphocreatine concentrations. At higher concentrations (3 and 10 μM), wortmannin completely blocked the contraction-stimulated glucose uptake but also decreased the contractility. In conclusion, inhibition of PI 3-kinase with wortmannin in skeletal muscle coincides with inhibition of insulin-stimulated glucose uptake and transport. Furthermore, in contrast to recent findings in incubated muscle, wortmannin also inhibited contraction-stimulated glucose uptake and transport. The inhibitory effect of wortmannin on contraction-stimulated glucose uptake may be independent of PI 3-kinase activity or due to inhibition of a subfraction of PI 3-kinase with low sensitivity to wortmannin.