Insulin provoked rapid increases in enzyme activity of immunoprecipitable protein kinase C-zeta (PKC-zeta) in rat adipocytes. Concomitantly, insulin provoked increases in 32P labeling of PKC-zeta both in intact adipocytes and during in vitro assay of immunoprecipitated PKC-zeta; the latter probably reflected autophosphorylation, as it was inhibited by the PKC-zeta pseudosubstrate. Insulin-induced activation of immunoprecipitable PKC-zeta was inhibited by LY294002 and wortmannin; this suggested dependence upon phosphatidylinositol (PI) 3-kinase. Accordingly, activation of PI 3-kinase by a pYXXM-containing peptide in vitro resulted in a wortmannin-inhibitable increase in immunoprecipitable PKC-zeta enzyme activity. Also, PI-3,4-(PO4)2, PI-3,4,5-(PO4)3, and PI-4,5-(PO4)2 directly stimulated enzyme activity and autophosphoralytion in control PKC-zeta immunoprecipitates to levels observed in insulin-treated PKC-zeta immunoprecipitates. In studies of glucose transport, inhibition of immunoprecipitated PKC-zeta enzyme activity in vitro by both the PKC-zeta pseudosubstrate and RO 31-8220 correlated well with inhibition of insulin-stimulated glucose transport in intact adipocytes. Also, in adipocytes transiently expressing hemagglutinin antigen-tagged GLUT4, co-transfection of wild-type or constitutive PKC-zeta stimulated hemagglutinin antigen-GLUT4 translocation, whereas dominant-negative PKC-zeta partially inhibited it. Our findings suggest that insulin activates PKC-zeta through PI 3-kinase, and PKC-zeta may act as a downstream effector of PI 3-kinase and contribute to the activation of GLUT4 translocation.