It has been shown that hyperglycaemia-induced defects in glucose transport and insulin action are mediated by increased flux of excess glucose through the hexosamine biosynthesis pathway (HBP). We have previously demonstrated that in rat adipocytes, increased flux through the HBP activates protein kinase C (PKC). The aim of the present study was to explore the mechanism for HBP-mediated activation of PKC. We show that activation of the HBP by either high glucose or glucosamine causes the translocation of PKC-zeta/lambda and PKC-epsilon but not other PKC isoforms tested (alpha, beta, delta). This translocation was inhibited by wortmannin, a PI 3-kinase inhibitor. Both high glucose and glucosamine caused widespread cellular activation of PI 3-kinase. We demonstrate that HBP-mediated activation of PI 3-kinase has an insulin-like effect to translocate GLUT4. We conclude that an acute increase of glucose flux through the HBP activates PI 3-kinase.