The effects of wortmannin, a selective inhibitor of phosphatidylinositol (PI) 3-kinase, on increases in glucose transport activity induced by insulin and norepinephrine (NE) have been studied in primary culture of brown adipocytes. Both insulin and NE produced 2- to 3-fold increase in 2-deoxy-D-[3H]glucose (2-DG) transport into cultured brown adipocytes. The effect of insulin was dependent upon translocation of the muscle/adipocyte glucose transporter (GLUT4) from microsome to plasma membranes. In contrast, NE did not affect the subcellular distribution of GLUT4. When wortmannin was added to the medium before stimulation with insulin, the increase in glucose transport and translocation of GLUT4 were completely abolished. On the other hand, NE-induced increase in glucose transport was resistant to wortmannin. These results indicate that PI 3-kinase plays a critical role on insulin-induced translocation of GLUT4, but that this pathway is not involved in the increase in glucose transport by NE in brown adipocytes.