To date there is suggestive evidence that phosphatidylinositol 3-kinase participates in insulin-stimulated glucose transport. However, its involvement in skeletal muscle, a major site of insulin-stimulated glucose disposal, has not been addressed. Therefore, we tested the effects of wortmannin, a known inhibitor of phosphatidylinositol 3-kinase, on insulin-stimulated 2-deoxyglucose uptake by rat soleus muscle in vitro. Wortmannin (1 μM) reversibly inhibited insulin-induced 2-deoxyglucose uptake in soleus muscle by 44%. Inclusion of 5 μM wortmannin in the incubation medium completely abolished the insulin-induced increment in 2-deoxyglucose uptake. In conclusion, the results are consistent with the concept that phosphatidylinositol 3-kinase plays a role in the insulin-signaling cascade linking insulin-receptor tyrosine kinase activation to glucose uptake in skeletal muscle.