Insulin stimulates glucose transport in muscle and fat cells by promoting the translocation of glucose transporter (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3-kinase) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt and PKC-ζ, those are known as the downstream target of PI3-kinase in regulation of GLUT4 translocation, is not known yet. An interesting possibility is that these protein kinases phosphorylate GLUT4 directly in this process. In the present study, PKB-α and PKC-ζ were added exogenously to GLUT4-containing vesicles purified from low density microsome (LDM) of the rat adipocytes by immunoadsorption and immunoprecipitation for direct phosphorylation of GLUT4. Interestingly GLUT4 was phosphorylated by PKC-ζ and its phosphorylation was increased in insulin stimulated state but GLUT4 was not phosphorylated by PKB-α. However, the GST-fusion proteins, GLUT4 C-terminal cytoplasmic domain (GLUT4C) and the entire major GLUT4 cytoplasmic domain corresponding to N-terminus, central loop and C-terminus in tandem (GLUT4NLC) were phosphorylated by both PKB-α and PKC-ζ. The immunoblots of PKC-ζ and PKB-α antibodies with GLUT4-containing vesicles preparation showed that PKC-ζ was co-localized with the vesicles but not PKB-α. From the above results, it is clear that PKC-ζ interacts with GLUT4-containing vesicles and it phosphorylates GLUT4 protein directly but PKB-α does not interact with GLUT4, suggesting that insulin-elicited signals that pass through PI3-kinase subsequently diverge into two independent pathways, an Akt pathway and a PKC-ζ pathway, and that later pathway contributes, at least in part, insulin stimulation of GLUT4 translocation in adipocytes via a direct GLUT4 phosphorylation.