The cellular receptor for hepatitis B virus (HBV) infection has not yet been identified. The purpose of this study was to address the possibility of participation by desialylated HBV and the asialoglycoprotein receptor (ASGP-R) exclusively expressed on liver parenchymal cells, in infection. Assays for viral binding and entry were performed by culturing a hepatoblastoma cell line, HepG2, and HBV particles derived from the HBV carrier in the presence or absence of neuraminidase (NA). Viral binding and entry were clearly enhanced in the presence of NA, and the enhancement of the binding could be blocked by asialo-fetuin and ethylenediamine-tetraacetic acid (EDTA). In addition, covalently closed circular (CCC)-DNA, as a marker of infectivity, was detected in the presence of NA, but not in its absence. The optimal concentration of NA raised infectivity more than 1000 times. We concluded that this method makes it feasible to evaluate the infectivity of HBV in vitro and that ASGP-R may be a specific HBV receptor once viral particles are desialylated.