Analysis of hepatitis B virus (HBV) infection has been difficult due to a lack of suitable culture system. Pseudotyped viruses provide a safe viral entry model because of their inability to produce infectious progeny virus. Here a system has been developed to prepare vesicular stomatitis virus (VSV) pseudotypes bearing various surface proteins of HBV(HBsAg): S, M or L. The infectivities of the psedotypes were determined by quantifying the number of cells expressing green fluorescent protein (GFP). The infectious unit (IU) of the pseudotype samples neutralized with anti-VSV in the absence and presence of anti-HBs S MAbs and titrated on HepG2 cells ranged from 1,000 to 4,000 IU/ml and 200 to 400 IU/ml, respectively. VSV pseudotype sample bearing HBs M antigen showed the highest infectious titer. This infectivity was inhibited by treatment with lactoferrin or sulfated polysachharides. Pretreatment of the cells with trypsin, tunicamycin or DTT also inhibited the infectivity of the pseudotypes. The VSVG*(HBV) pseudotypes might be useful as a safe and rapid assay system to clarify early steps of HBV infection.