Hepatitis B virus-infected patients secrete enormous quantities (50-300 microg/ml) of hepatitis B surface antigen (HBsAg) in their serum. One hypothesis for this synthetic effort is that these lipoprotein particles serve to adsorb neutralizing antisurface antibodies. We have shown that insulin suppresses the expression of HBsAg in human hepatoma cell Hep3B cells. We further studied the signaling pathway of insulin on the inhibition of HBsAg. Using a fungal metabolite, lovastatin, to block the p21Ras signaling pathway of insulin, we found that lovastatin inhibited the secretion of HBsAg into culture medium in Hep3B cells; however, the involvement of p21Ras-MAPKs was excluded in this effect. The cholesterol depletion from the membrane, leading to the destabilization of rafts, was the mechanism for the lovastatin inhibition of HBsAg secretion. However, lovastatin has no effect on the secretion of infectious viral Dane particles. Herein, we show for the first time that cholesterol is required for HBsAg secretion.