The aim of this study was to examine the effects of insulin and phorbol 12-myristate 13-acetate (PMA), an activator of classic and novel PKCs, on the translocation of PKC from cytosol to membrane in H4IIE (H4) rat hepatoma cells. Six PKC isoforms were expressed, including PKC-mu and PKC-lambda, identified for the first time in this hepatoma-cell line. Insulin induced translocation of PKC-delta from the cytosol to the membrane fraction as early as 15 min and maximally at 60 min with levels returning to that of controls by 180 min. Insulin also decreased levels of PKC-zeta in membranes at 5, 10, 15, and 30 min, but had no effect on cytosol levels. Ten minutes of PMA treatment translocated PKC-delta completely, and 24 h of PMA treatment downregulated PKC-delta. Neither acute nor chronic PMA had any effect on PKC-zeta. These studies establish the ability of both insulin and PMA to activate PKC-delta in H4 cells, and coupled with our previous work demonstrating a diminution of the effect of insulin on gene transcription in PKC downregulated cells, suggest that insulin may exert specific effects, in part, through a PKC-dependent pathway.