Both the anabolic hormone insulin and contractile activity stimulate the uptake of glucose into mammalian skeletal muscle. In this study, we examined the role of phosphatidylinositol 3-kinase (PI 3-kinase), a putative mediator of insulin actions, in the stimulation of hexose uptake in response to hormone and contraction. Phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate accumulate in skeletal muscle exposed to insulin but not hypoxia, which mimics stimulation of the contractile-dependent pathway of hexose transport activation. The fungal metabolite wortmannin, an inhibitor of PI 3-kinase, completely blocks the appearance of 3'-phospholipids in response to insulin. Moreover, wortmannin entirely prevented the increase in hexose uptake in muscle exposed to insulin but was without effect on muscle stimulated by repetitive contraction or hypoxia. These results support the view that PI 3-kinase is involved in the signaling pathways mediating insulin-responsive glucose transport in skeletal muscle but is not required for stimulation by hypoxia or contraction. Furthermore, these data indicate that there exist at least two signaling pathways leading to activation of glucose transport in skeletal muscle with differential sensitivities to wortmannin.