Binding of viral envelope proteins to specific receptors on human hepatocytes is considered to be an important step in HBV infection. In this study, we demonstrate that a 34-kDa human liver plasma membrane protein specifically binds to small HBsAg in a Ca(2+)-dependent manner. By partial amino acid sequence analysis of preparatively isolated 34-kDa protein comigrating with HBsAg-binding protein obtained from binding assay on IEF/SDS-PAGE, we have identified this HBsAg-binding protein as Endonexin II (E-II). Native human liver E-II inhibits binding of HBsAg to intact human hepatocytes and shows specific binding to small HBsAg. This binding can be inhibited by human liver plasma membrane proteins, recombinant E-II, or anti-E-II antibodies. Despite 90% sequence homology, rat liver E-II does not bind to small HBsAg and does not inhibit significantly (less than 20%) binding of HBsAg to intact hepatocytes. Cross-linking of small HBsAg and radiolabeled human liver E-II resulted in a specific additional protein complex on PAGE with an apparent molecular weight of 90 kDa, corresponding to a complex of E-II and small HBsAg with a ratio of 2 to 1 or 1 to 2. These findings indicate that E-II, found in human liver, is a specific HBsAg-binding protein and might play an important role in the initiation of HBV infection.