Insulin suppresses hepatitis B surface antigen (HBsAg) gene expression and stimulates cell proliferation in human hepatoma Hep3B cells. 12-O-tetradecanoyl phorbol-13-acetate, TPA, has been demonstrated to mimic insulin actions in these cells. We examined the role of phosphatidylinositol 3-kinase (PI 3-kinase) in the signaling pathways of insulin and TPA towards these two biological phenomena in Hep3B cells. The pre-treatment of 5 microM of wortmannin diminished insulin suppressed HBsAg production and completely abolished insulin stimulated cell proliferation. However, wortmannin had no effect on TPA actions in both HBsAg suppression and cell growth stimulation. We further investigated the effect of wortmannin in mitogen-activated protein kinases (MAPKs) activation induced by insulin or TPA. After the pretreatment of wortmannin, insulin activated MAPKs was completely blocked, but TPA was still capable to activate MAPKs. These results suggest that PI 3-kinase is involved in insulin actions but not in TPA effects, and allow us to dissociate the signaling pathways of insulin and TPA in human hepatoma Hep3B cells.