We examined the question of whether insulin activates protein kinase C (PKC)-ζ in L6 myotubes, and the dependence of this activation on phosphatidylinositol (PI) 3-kinase. We also evaluated a number of issues that are relevant to the question of whether diacylglycerol (DAG)-dependent PKCs or DAG-insensitive PKCs, such as PKC-ζ, are more likely to play a role in insulin-stimulated glucose transport in L6 myotubes and other insulin-sensitive cell types. We found that insulin increased the enzyme activity of immunoprecipitable PKC-ζ in L6 myotubes, and this effect was blocked by PI 3-kinase inhibitors, wortmannin and LY294002; this suggested that PKC-ζ operates downstream of PI 3-kinase during insulin action. We also found that treatment of L6 myotubes with 5 μm tetradecanoyl phorbol-13-acetate (TPA) for 24 h led to 80–100% losses of all DAG-dependent PKCs (α, β₁, β₂, δ, ε) and TPA-stimulated glucose transport (2-deoxyglucose uptake); in contrast, there was full retention of PKC-ζ, as well as insulin-stimulated glucose transport and translocation of GLUT4 and GLUT1 to the plasma membrane. Unlike what has been reported in BC3H-1 myocytes, TPA treatment did not elicit increases in PKCβ2 messenger RNA or protein in L6 myotubes, and selective retention of this PKC isoform could not explain the retention of insulin effects on glucose transport after prolonged TPA treatment. Of further interest, TPA acutely activated membrane-associated PI 3-kinase in L6 myotubes, and acute effects of TPA on glucose transport were inhibited, not only by the PKC inhibitor, LY379196, but also by both wortmannin and LY294002; this suggested that DAG-sensitive PKCs activate glucose transport through cross-talk with phosphatidylinositol (PI) 3-kinase, rather than directly through PKC. Also, the cell-permeable, myristoylated PKC-ζ pseudosubstrate inhibited insulin-stimulated glucose transport both in non-down-regulated and PKC-depleted (TPA-treated) L6 myotubes; thus, the PKC-ζ pseudosubstrate appeared to inhibit a protein kinase that is required for insulin-stimulated glucose transport but is distinct from DAG-sensitive PKCs. In keeping with the latter dissociation of DAG-sensitive PKCs and insulin-stimulated glucose transport, LY379196, which inhibits PKC-β (preferentially) and other DAG-sensitive PKCs at relatively low concentrations, inhibited insulin-stimulated glucose transport only at much higher concentrations, not only in L6 myotubes, but also in rat adipocytes, BC3H-1 myocytes, 3T3/L1 adipocytes and rat soleus muscles. Finally, stable and transient expression of a kinase-inactive PKC-ζ inhibited basal and insulin-stimulated glucose transport in L6 myotubes. Collectively, our findings suggest that, whereas PKC-ζ is a reasonable candidate to participate in insulin stimulation of glucose transport, DAG-sensitive PKCs are unlikely participants.